Human ADAMTS-1 protein, gene encoding the same, pharmaceutical composition, and method for immunologically analyzing human ADAMTS-1 protein

ABSTRACT

A human ADAMTS-1 protein, a gene encoding the same, a pharmaceutical composition containing the protein as an active ingredient, and a method for immunologically analyzing the human ADAMTS-1 protein are disclosed. The protein can decrease the number of leukocytes and platelets, and at the same time, increase the number of erythrocytes.

TECHNICAL FIELD

[0001] The present invention relates to a human ADAMTS-1 protein, a geneencoding the same, a pharmaceutical composition, and a method forimmunologically analyzing the human ADAMTS-1 protein.

BACKGROUND ART

[0002] A mouse ADAMTS (A disintegrin and metalloproteinase withthrombospondin motifs)-1 gene has been cloned as a cDNA from a mousecolon cancer cell which induces cancer cachexia when transplanted to amouse. The mouse ADAMTS-1 protein encoded by the gene is a uniqueprotein containing a matrix metalloproteinase domain, a disintegrindomain, and three thrombospondin domains [J. Biol. Chem., 272, 556-562(1997)]. The physiological functions of the mouse ADAMTS-1 protein hasnot been reported, but there have been reports of each individualfunctional domain contained therein.

[0003] For example, a snake venom disintegrin belongs to a family ofproteins which are rich in cystein, and exhibit an anticoagulantactivity [Semin. Hematol., 31, 289-300 (1994)].

[0004] Further, for example, an ADAM (A disintegrin andmetalloproteinase) family is known as a protein family containing amatrix metalloproteinase domain and a disintegrin domain [Nature, 377,652-656 (1995); Nature Genet., 5, 151-157 (1993); Nature, 356, 248-252(1992)]. Examples of known ADAM family proteins are fertilin, epidermalapical protein, cyritestin, MDC (metalloprotease-like, disintegrin-likeand cystein-rich protein), meltrin, MS2, and metargidin [Nature, 377,652-656 (1995); Nature Genet. 5, 151-157 (1993); Nature, 356, 248-252(1992); Biochem. J., 286, 671-675 (1992); Dev. Growth. Differ., 36,49-58 (1994); Int. Immunol., 2, 585-591 (1990); J. Biol. Chem., 271,4593-4596 (1996)].

[0005] It was reported that fertilin is involved in an integrin-mediatedsperm-egg binding [Nature, 356, 248-252 (1992)], and meltrin is involvedin a myotube formation [Nature, 377, 652-656 (1995)]. MDC expressedmainly in a central nervous system is a candidate as a suppresseragainst a human breast cancer [Nature Genet. 5, 151-157 (1993)], and MS2serves as a macrophage antigen [Int. Immunol., 2, 585-591 (1990)].However, little have been known about the physiological roles of theseADAM family proteins.

[0006] The mouse ADAMTS-1 protein contains a matrix metalloproteinasedomain and a disintegrin domain, and therefore belongs to the ADAMfamily. However, the mouse ADAMTS-1 protein is different from otherknown ADAM family proteins in that it further contains thrombospondindomains.

[0007] As mentioned, the ADAM family proteins have various kinds ofactivities such as the involvement of bone or muscle metabolism,suppression of cancer growth, or fertilization, and the thrombospondinexhibits an action to inhibit vascularization and suppresses cancer.Therefore, it is expected that the mouse ADAMTS-1 protein will exhibitunique physiological functions.

[0008] The inventors of the present invention attempted to isolate thecorresponding human ADAMTS-1 protein. Accordingly, the present inventorsdesigned and prepared various probes on the basis of the base sequenceof the known mouse ADAMTS-1 gene, and carried out plaque hybridizationswith a human kidney cDNA library so as to obtain a human ADAMTS-1 gene,but a desired gene was not obtained. Then, the present inventorsdesigned and prepared various primers on the basis of the base sequenceof the known mouse ADAMTS-1 gene, and carried out PCRs, using the humankidney cDNA library as templates under ordinary conditions, so as toobtain the desired gene, but did not succeed.

[0009] Thereafter, the present inventors carried out a PCR of the humankidney cDNA library, using the same primers, but under conditions milderthan those ordinarily used, more particularly, an annealing temperatureis set lower than an ordinary temperature, and the inventorssuccessfully obtained a novel human ADAMTS-1 gene. The resulting genewas then, expressed in E. coli, and the biological activities of therecombinant human ADAMTS-1 protein were examined. Surprisingly, it wasfound that the novel human ADAMTS-1 protein can decrease the numbers ofleukocytes and platelets, and at the same time, increase the number oferythrocytes. Such activities in influencing hematopoietic functionscannot be expected from the structure of the mouse ADAMTS-1 gene whichwas used as the basis in designing the primers, or from the functions ofdomains contained in the human ADAMTS-1 protein. The present inventionis based on the above findings.

DISCLOSURE OF INVENTION

[0010] The present invention relates to a protein characterized bycontaining an amino acid sequence of SEQ ID NO: 1: Met Asp Ile Cys ArgIle Arg Leu Arg Lys Lys Arg Phe Val Ser Ser Pro Arg Tyr Val Glu Thr MetLeu Val Ala Asp Gln Ser Met Ala Glu Phe His Gly Ser Gly Leu Lys His TyrLeu Leu Thr Leu Phe Ser Val Ala Ala Arg Leu Tyr Lys His Pro Ser Ile ArgAsn Ser Val Ser Leu Val Val Val Lys Ile Leu Val Ile His Asp Glu Gln LysGly Pro Glu Val Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn TrpGln Lys Gln His Asn Pro Pro Ser Asp Arg Asp Ala Glu His Tyr Asp Thr AlaIle Leu Phe Thr Arg Gln Asp Leu Cys Gly Ser Gln Thr Cys Asp Thr Leu GlyMet Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser Cys Ser Val Ile GluAsp Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala His Glu Leu Gly His Val PheAsn Met Pro His Asp Asp Ala Lys Gln Cys Ala Ser Leu Asn Gly Val Asn GlnAsp Ser His Met Met Ala Ser Met Leu Ser Asn Leu Asp His Ser Gln Pro TrpSer Pro Cys Ser Ala Tyr Met Ile Thr Ser Phe Leu Asp Asn Gly His Gly GluCys Leu Met Asp Lys Pro Gln Asn Pro Ile Gln Leu Pro Gly Asp Leu Pro GlyThr Leu Tyr Asp Ala Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu Asp Ser LysHis Cys Pro Asp Ala Ala Ser Thr Cys Ser Thr Leu Trp Cys Thr Gly Thr SerGly Gly Val Leu Val Cys Gln Thr Lys His Phe Pro Trp Ala Asp Gly Thr SerCys Gly Glu Gly Lys Trp Cys Ile Asn Gly Lys Cys Val Asn Lys Thr Asp ArgLys His Phe Asp Thr Pro Phe His Gly Ser Trp Gly Pro Trp Gly Pro Trp GlyAsp Cys Ser Arg Thr Cys Gly Gly Gly Val Gln Tyr Thr Met Arg Glu Cys AspAsn Pro Val Pro Lys Asn Gly Gly Lys Tyr Cys Glu Gly Lys Arg Val Arg TyrArg Ser Cys Asn Leu Glu Asp Cys Pro Asp Asn Asn Gly Lys Thr Phe Arg GluGlu Gln Cys Glu Ala His Asn Glu Phe Ser Lys Ala Ser Phe Gly Ser Gly ProAla Val Glu Trp Ile Pro Lys Tyr Ala Gly Val Ser Pro Lys Asp Arg Cys LysLeu Ile Cys Gln Ala Lys Gly Ile Gly Tyr Phe Phe Val Leu Gln Pro Lys ValVal Asp Gly Thr Pro Cys Ser Pro Asp Ser Thr Ser Val Cys Val Gln Gly GlnCys Val Lys Ala Gly Cys Asp Arg Ile Ile Asp Ser Lys Lys Lys Phe Asp LysCys Gly Val Cys Gly Gly Asn Gly Ser Thr Cys Lys Lys Ile Ser Gly Ser ValThr Ser Ala Lys Pro Gly Tyr His Asp Ile Val Thr Ile Pro Thr Gly Ala ThrAsn Ile Glu Val Lys Gln Arg Asn Gln Arg Gly Ser Arg Asn Asn Gly Ser PheLeu Ala Ile Lys Ala Ala Asp Gly Thr Tyr Ile Leu Asn Gly Asp Tyr Thr LeuSer Thr Leu Glu Gln Asp Ile Met Tyr Lys Gly Val Val Leu Arg Tyr Ser GlySer Ser Ala Ala Leu Glu Arg Ile Arg Ser Phe Ser Pro Leu Lys Glu Pro LeuThr Ile Gln Val Leu Thr Val Gly Asn Ala Leu Arg Pro Lys Ile Lys Tyr ThrTyr Phe Val Lys Lys Lys Lys Glu Ser Phe Asn Ala Ile Pro Thr Phe Ser AlaTrp Val Ile Glu Glu Trp Gly Glu Cys Ser Lys Ser Cys Glu Leu Gly Trp GlnArg Arg Leu Val Glu Cys Arg Asp Ile Asn Gly Gln Pro Ala Ser Glu Cys AlaLys Glu Val Lys Pro Ala Ser Thr Arg Pro Cys Ala Asp His Pro Cys Pro GlnTrp Gln Leu Gly Glu Trp Ser Ser Cys Ser Lys Thr Cys Gly Lys Gly Tyr LysLys Arg Ser Leu Lys Cys Leu Ser His Asp Gly Gly Val Leu Ser His Glu SerCys Asp Pro Leu Lys Lys Pro Lys His Phe Ile Asp Phe Cys Thr Leu Thr GlnCys Ser.

[0011] Further, the present invention relates to the variation ofproteins which are equivalent to the protein containing the amino acidsequence of SEQ ID NO: 1.

[0012] Further, the present invention relates to a protein characterizedby containing a matrix metalloproteinase domain, a disintegrin domain,and a thrombospondin domain, except for a mouse ADAMS-1 protein.

[0013] Further, the present invention relates to a gene characterized byencoding the above novel proteins.

[0014] Further, the present invention relates to a vector characterizedby containing the above gene.

[0015] Further, the present invention relates to a transformantcharacterized by being transformed by the above vector.

[0016] Further, the present invention relates to a pharmaceuticalcomposition characterized by comprising (1) the protein containing theamino acid sequence of SEQ ID NO: 1, (2) the variation functionallyequivalent to the protein containing the amino acid sequence of SEQ IDNO: 1, or (3) the protein containing a matrix metalloproteinase domain,a disintegrin domain, and a thrombospondin domain.

[0017] Further, the present invention relates to an immunologicallyreactive substance (such as a polyclonal antibody or a monoclonalantibody, or an antibody fragment thereof, or an antiserum)characterized by nature of being capable of specifically reacting withthe novel proteins.

[0018] Further, the present invention relates to a method forimmunologically analyzing the human ADAMTS-1 protein, characterized inthat a sample is brought into contact with the immunologically reactivesubstance, and a complex of the human ADAMTS-1 protein and theimmunologically reactive substance is detected.

[0019] Further, the present invention relates to a method for analyzingan mRNA of the human ADAMTS-1 protein, characterized in that a sample isbrought into contact with a polynucleotide containing a base sequencecomplementary to that of the mRNA of the human ADAMTS-1 proteinconsisting of the amino acid sequence of SEQ ID NO: 1, and a complex ofthe mRNA of the human ADAMTS-1 protein and the gene is detected.

[0020] Further, the present invention relates to a method forextracorporeally detecting an immunological state, characterized byanalyzing the human ADAMTS-1 protein or the mRNA thereof.

[0021] Further, the present invention relates to an agent for analyzingan immunological state, characterized by containing the immunologicallyreactive substance capable of immunologically reacting the humanADAMTS-1 protein or the polynucleotide containing the base sequencecomplementary to that of the mRNA of the human ADAMTS-1 protein.

BRIEF DESCRIPTION OF DRAWINGS

[0022]FIG. 1 shows the results of an electrophoresis of a Flag. 1 DNAfragment produced by a PCR.

[0023]FIG. 2 shows a homology between the mouse ADAMTS-1 gene and theFlag. 1 DNA fragment.

[0024]FIG. 3 shows results of dot hybridization of the Flag. 1 DNAfragment.

[0025]FIG. 4 shows results of an electrophoresis of a Flag. 2 DNAfragment produced by a RACE.

[0026]FIG. 5 shows a homology in base sequences of the bases 1 to 480 inthe human ADAMTS-1 gene and the mouse ADAMTS-1 gene.

[0027]FIG. 6 shows a homology in base sequences of the bases 481 to 960in the human ADAMTS-1 gene and the mouse ADAMTS-1 gene.

[0028]FIG. 7 shows a homology in base sequences of the bases 961 to 1440between the human ADAMTS-1 gene and the mouse ADAMTS-1 gene.

[0029]FIG. 8 shows a homology in base sequences of the bases 1441 to1920 between the human ADAMTS-1 gene and the mouse ADAMTS-1 gene.

[0030]FIG. 9 shows a homology in base sequences of the bases 1921 to2184 between the human ADAMTS-1 gene and the mouse ADAMTS-1 gene.

[0031]FIG. 10 shows a homology in amino acid sequences of the aminoacids 1 to 240 between the human ADAMTS-1 protein and the mouse ADAMTS-1protein.

[0032]FIG. 11 shows a homology in amino acid sequences of the aminoacids 241 to 510 between the human ADAMTS-1 protein and the mouseADAMTS-1 protein.

[0033]FIG. 12 shows a homology in amino acid sequences of the aminoacids 511 between 727 in the human ADAMTS-1 protein and the mouseADAMTS-1 protein.

[0034]FIG. 13 shows results of an electrophoresis of a full-length cDNAof the human ADAMTS-1 gene of the present invention, the cDNA beingproduced by a PCR.

[0035]FIG. 14 schematically illustrates a structure of plasmidpG/ADAMTS-1 of the present invention.

[0036]FIG. 15 shows results of an electrophoresis of a transformanttransformed by the plasmid pG/ADAMTS-1.

[0037]FIG. 16 shows results of an electrophoresis of a GST-humanADAMTS-1 fusion protein.

[0038]FIG. 17 provides graphs showing effects on the numbers of bloodcells when the GST-human ADAMTS-1 fusion protein is intravenouslyadministered to a mouse in a single dosage.

BEST MODE FOR CARRYING OUT THE INVENTION

[0039] The present invention will be explained in detail hereinafter.

[0040] The human ADAMTS-1 protein of the present invention is a novelprotein consisting of 727 amino acid residues; that is, consisting ofthe amino acid sequence of SEQ ID NO: 1. As shown in FIGS. 10 to 12, thehuman ADAMTS-1 protein of the present invention contains a matrixmetalloproteinase (hereinafter sometimes referred to as “MMP”) domainconsisting of the 12th to 230th amino acid residues counting from theN-terminal amino acid residue, methionine; a disintegrin (hereinaftersometimes referred to as “DI”) domain consisting of the 235th to 305thamino acid residues; and three thrombospondin (hereinafter sometimesreferred to as “TSP”) domains consisting of the 322nd to 372nd, 618th to664th, and 672nd to 727th amino acid residues. The human ADAMTS-1protein contains many arginines and lysines, which are basic aminoacids, in a C-terminal region. Therefore, it is believed that the humanADAMTS-1 protein interacts with sulfated polysaccharide molecules, suchas heparin or heparan sulfate, in blood.

[0041] The novel protein according to the present invention includes theprotein containing the amino acid sequence of SEQ ID NO: 1; and avariation functionally equivalent to the protein containing the aminoacid sequence of SEQ ID NO: 1 (hereinafter sometimes referred to as a“human ADAMTS-1 protein variation”). The term “human ADAMTS-1 proteinvariation” as used herein means a protein having an amino acid sequencewherein one or more (particularly one or several) amino acids aredeleted in, changed in, or inserted to the amino acid sequence of thehuman ADAMTS-1 protein, that is, the amino acid sequence of SEQ ID NO:1, and exhibiting activities of the human ADAMTS-1 protein. A preferablehuman ADAMTS-1 protein variation has a 92% or more homology in the aminoacid sequence with the human ADAMTS-1 protein. The human ADAMTS-1protein variation includes a fragment which is a part of the proteincontaining the amino acid sequence of SEQ ID NO: 1 and exhibits theactivities of the human ADAMTS-1 protein; and a fragment which is a partof another human ADAMTS-1 protein variation and exhibits the activitiesof the human ADAMTS-1 protein.

[0042] The term “human ADAMTS-1 activity” as used herein means anactivity to decrease the numbers of leukocytes and platelets, and at thesame time, increase the number of erythrocytes.

[0043] Further, the novel protein of the present invention includes aprotein containing a matrix metalloproteinase domain, a disintegrindomain, and a thrombospondin domain (hereinafter sometimes referred toas an “ADAMTS protein”). However, the mouse ADAMTS-1 protein is notincluded in the novel protein of the present invention.

[0044] The term “matrix metalloproteinase domain” as used herein means adomain containing an amino acid sequence having a 50% or more(preferably 95% or more) homology with the amino acid sequence of thematrix metalloproteinase domain in the human ADAMTS-1 protein, i.e., theamino acid sequence of the 12th to 230th amino acids in the amino acidsequence of SEQ ID NO: 1.

[0045] The term “disintegrin domain” as used herein means a domaincontaining an amino acid sequence having a 50% or more (preferably 93%or more) homology with the amino acid sequence of the disintegrin domainin the human ADAMTS-1 protein, i.e., the amino acid sequence of the235th to 305th amino acids in the amino acid sequence of SEQ ID NO: 1.

[0046] The term “thrombospondin domain” as used herein means a domaincontaining an amino acid sequence having a 50% or more homology with atleast one of the amino acid sequences of three disintegrin domains inthe human ADAMTS-1 protein, that is,

[0047] (1) a domain containing an amino acid sequence having a 50% ormore (preferably 99% or more) homology with the amino acid sequence ofthe first thrombospondin domain (hereinafter sometimes referred to as a“human TSP-1 domain”) from the N-terminus in the human ADAMTS-1 protein,i.e., the amino acid sequence of the 322nd to 372nd amino acids in theamino acid sequence of SEQ ID NO: 1;

[0048] (2) a domain containing an amino acid sequence having a 50% ormore (preferably 88% or more) homology with the amino acid sequence ofthe second thrombospondin domain (hereinafter sometimes referred to as a“human TSP-2 domain”) from the N-terminus in the human ADAMTS-1 protein,i.e., the amino acid sequence of the 618th to 664th amino acids in theamino acid sequence of SEQ ID NO: 1; or

[0049] (3) a domain containing an amino acid sequence having a 50% ormore (preferably 88% or more) homology with the amino acid sequence ofthe third thrombospondin domain (hereinafter sometimes referred to as a“human TSP-3 domain”) from the N-terminus in the human ADAMTS-1 protein,i.e., the amino acid sequence of the 672nd to 727th amino acids in theamino acid sequence of SEQ ID NO: 1.

[0050] In the ADAMTS protein of the present invention, the number ofeach of the matrix metalloproteinase domain, the disintegrin domain, andthe thrombospondin domain, and the sequential order thereof, are notparticularly limited, so long as at least one matrix metalloproteinasedomain, at least one disintegrin domain, and at least one thrombospondindomain are contained at the same time in the ADAMTS protein. A preferredADAMTS protein contains a matrix metalloproteinase domain, a disintegrindomain and three thrombospondin domains. Further, the sequential orderof the domains from the N-terminus to the C-terminus preferably startsfrom the matrix metalloproteinase domain, followed by the disintegrindomain, and then the thrombospondin domain. When three thrombospondindomains are contained, the sequential order of the domains from theN-terminus to the C-terminus preferably starts from the matrixmetalloproteinase domain, followed by the disintegrin domain, and thenthe first TSP domain, the second TSP domain and the third TSP domain.

[0051] In the ADAMTS protein of the present invention, it is preferablethat

[0052] (1) the matrix metalloproteinase domain has a 95% or morehomology in the amino acid sequences with the matrix metalloproteinasedomain in the human ADAMTS-1 protein,

[0053] (2) the disintegrin domain has a 93% or more homology in theamino acid sequences with the disintegrin domain in the human ADAMTS-1protein, and

[0054] (3) at least one of the thrombospondin domains has a 99% or morehomology in the amino acid sequence with the TSP-1 domain in the humanADAMTS-1 protein, an 88% or more homology in the amino acid sequenceswith the TSP-2 domain in the human ADAMTS-1 protein, or an 88% or morehomology in the amino acid sequences with the TSP-3 domain in the humanADAMTS-1 protein.

[0055] In the present ADAMTS protein containing three thrombospondindomains, it is preferable that

[0056] (3-1) the first thrombospondin domain from the N-terminus has a99% or more homology in the amino acid sequences with the TSP-1 domainin the human ADAMTS-1 protein,

[0057] (3-2) the second thrombospondin domain from the N-terminus has an88% or more homology in the amino acid sequences with the TSP-2 domainin the human ADAMTS-1 protein, and

[0058] (3-3) the third thrombospondin domain from the N-terminus has an88% or more homology in the amino acid sequences with the TSP-3 domainin the human ADAMTS-1 protein.

[0059] The protein of the present invention may be prepared by variousknown processes. For example, the protein of the present invention maybe prepared using a known genetic engineering technique and the gene ofthe present invention. Alternatively, the protein of the presentinvention may be purified from a naturally occurring source, using aknown protein chemical technique.

[0060] The gene of the present invention includes a gene encoding theprotein containing the amino acid sequence of SEQ ID NO: 1, particularlythe human ADAMTS-1 protein; a gene encoding the human ADAMTS-1 proteinvariation; and a gene encoding the ADAMTS protein (except the mouseADAMTS-1 protein). The present gene may be DNA or RNA. The gene encodingthe human ADAMTS-1 protein may be, for example, a gene consisting of thebase sequence of SEQ ID NO: 2: ATG GAT ATC TGC AGA ATT CGG CTT AGG AAGAAG CGA TTT GTG TCC AGC CCC CGT TAT GTG GAA ACC ATG CTT GTG GCA GAC CAGTCG ATG GCA GAA TTC CAC GGC AGT GGT CTA AAG CAT TAC CTT CTC ACG TTG TTTTCG GTG GCA GCC AGA TTG TAC AAA CAC CCC AGC ATT CGT AAT TCA GTT AGC CTGGTG GTG GTG AAG ATC TTG GTC ATC CAC GAT GAA CAG AAG CCG GGG GAA GTG ACCTCC AAT GCT GCC CTC ACT CTG CGG AAC TTT TGC AAC TGG CAG AAG CAG CAC AACCCA CCC AGT GAC CGG GAT GCA GAG CAC TAT GAC ACA GCA ATT CTT TTC ACC AGACAG GAC TTG TGT GGG TCC CAG ACA TGT GAT ACT CTT GGG ATG GCT GAT GTT GGAACT GTG TGT GAT CCG AGC AGA AGC TGC TCC GTC ATA GAA GAT GAT GGT TTA CAAGCT GCC TTC ACC ACA GCC CAT GAA TTA GGC CAC GTG TTT AAC ATG CCA CAT GATGAT GCA AAG GAG TGT GCC AGC CTT AAT GGT GTG AAC CAG GAT TCC CAC ATG ATGGCG TCA ATG CTT TCC AAC CTG GAC CAC AGC CAG CCT TGG TCT CCT TGC AGT GCCTAC ATG ATT ACA TCA TTT CTG GAT AAT GGT CAT GGG GAA TGT TTG ATG GAC AAGCCT CAG AAT CCC ATA CAG CTC CCA GGC GAT CTC CCT GGC ACC TTG TAC GAT GCCAAC CGG CAG TGC CAG TTT ACA TTT GGG GAG GAC TCC AAA CAC TGC CCC GAT GCAGCC AGC ACA TGT AGC ACC TTG TGG TGT ACC GGC ACC TCT GGT GGG GTG CTG GTGTGT CAA ACC AAA CAC TTC CCG TGG GCG GAT GGC ACC AGC TGT GGA GAA GGG AAATGG TGT ATC AAC GGC AAG TGT GTG AAC AAA ACC GAC AGG AAG CAT TTT GAT ACGCCT TTT CAT GGA AGC TGG GGA CCA TGG GGA CCG TGG GGA GAC TGT TCG AGA ACGTGC GGT GGA GGA GTC CAG TAC ACG ATG AGG GAA TGT GAC AAC CCA GTC CCA AAGAAT GGA GGG AAG TAC TGT GAA GGC AAA CGA GTG CGC TAC AGA TCC TGT AAC CTTGAG GAC TGT CCA GAC AAT AAT GGA AAA ACC TTT AGA GAG GAA CAA TGT GAA GCACAC AAC GAG TTT TCA AAA GCT TCC TTT GGG AGT GGG CCT GCG GTG GAA TGG ATTCCC AAG TAC GCT GGC GTC TCA CCA AAG GAC AGG TGC AAG CTC ATC TGC CAA GCCAAA GGC ATT GGC TAC TTC TTC GTT TTG CAG CCC AAG GAT GAT GGT GGT ACT CCATGT AGC CCA GAT TCC ACC TCT GTC TGT GTG CAA GGA CAG TGT GTA AAA GCT GGTTGT GAT CGC ATC ATA GAC TCC AAA AAG AAG TTT GAT AAA TGT GGT GTT TGC GGGGGA AAT GGA TCT ACT TGT AAA AAA ATA TCA GGA TCA GTT ACT AGT GCA AAA CCTGGA TAT CAT GAT ATC GTC ACA ATT CCA ACT GGA GCC ACC AAC ATC GAA GTG AAACAG CGG AAC CAG AGG GGA TCC AGG AAC AAT GGC AGC TTT CTT GCC ATC AAA GCTGCT GAT GGC ACA TAT ATT CTT AAT GGT GAC TAC ACT TTG TCC ACC TTA GAG CAAGAC ATT ATG TAC AAA GGT GTT GTC TTG AGG TAC AGC GGC TCC TCT GCG GCA TTGGAA AGA ATT CGC AGC TTT AGC CCT CTC AAA GAG CCC TTG ACC ATC CAG GTT CTTACT GTG GGC AAT GCC CTT CGA CCT AAA ATT AAA TAC ACC TAC TTC GTA AAG AAGAAG AAG GAA TCT TTC AAT GCT ATC CCC ACT TTT TCA GCA TGG GTC ATT GAA GAGTGG GGC GAA TGT TCT AAG TCA TGT GAA TTG GGT TGG CAG AGA AGA CTG GTA GAATGC CGA GAC ATT AAT GGA CAG CCT GCT TCC GAG TGT GCA AAG GAA GTG AAG CCAGCC AGC ACC AGA CCT TGT GCA GAC CAT CCC TGC CCC CAG TGG CAG CTG GGG GAGTGG TCA TCA TGT TCT AAG ACC TGT GGG AAG GGT TAC AAA AAA AGA AGC TTG AAGTGT CTG TCC CAT GAT GGA GGG GTG TTA TCT CAT GAG AGC TGT GAT CCT TTA AAGAAA CCT AAA CAT TTC ATA GAC TTT TGC ACA CTG ACA CAG TGC AGT TAA

[0061] The gene of the present invention, such as the gene consisting ofthe base sequence of SEQ ID NO: 2, may be obtained, for example, by thefollowing method, which was used by the present inventors when thepresent gene was obtained for the first time.

[0062] That is, various suitable primers for a PCR are prepared on thebasis of the base sequence of the mouse ADAMTS-1 gene. DNA fragments maybe obtained by carrying out a PCR under a condition milder than that ofan ordinary PCR, i.e., at an annealing temperature lower than a usualannealing temperature, using a human kidney cDNA library as templates.Base sequences of the resulting DNA fragments are determined, andcompared with the base sequence of the mouse ADAMTS-1 gene, to therebyidentify the resulting DNA fragments as the desired genes. Depending onthe primers used in the PCR, a full-length human ADAMTS-1 gene or apartial base sequence of the human ADAMTS-1 gene may be obtained. Whenthe partial base sequence is obtained, the remaining base sequence ofthe gene may be obtained by a RACE (Rapid amplification of cDNA ends)method [Proc Natl. Acad. Sci. USA, 85, 8998-9002 (1988)], and partialbase sequences may be ligated by genetic-engineering to obtain thefull-length base sequence.

[0063] When the present inventors designed the base sequences of theprimers used to obtain the gene in the above method for the first time,the base sequence of the human ADAMTS-1 gene was not known. Therefore,it was almost impossible to select base sequences having a completehomology between the mouse ADAMTS-1 gene and the human ADAMTS-1 gene.

[0064] The present inventors used primers designed on the basis of thebase sequence of the mouse ADAMTS-1 gene to carry out a PCR at a usualtemperature, but a desired DNA fragment could not be obtained. However,the present inventors used the same primers and carried out a PCR undera condition milder than that of an ordinary PCR, i.e., an annealingtemperature was set lower than a usual annealing temperature, wherebythe desired DNA fragment was able to be obtained. The comparison of thebase sequence of the resulting human ADAMTS-1 gene with the basesequences of the primers used revealed an insufficient homology.

[0065] At present, the base sequence of the human ADAMTS-1 gene has beendetermined according to the present invention. Therefore, the basesequence of the human ADAMTS-1 gene may be used to design primers for aPCR or probes for plaque hybridization. Such primers or probes may beused to obtain the gene of the present invention by a known method toobtain a gene, such as a PCR under ordinary conditions or plaquehybridization, instead of the method used by the present inventors toobtain the present gene for the first time.

[0066] The present inventors attempted to obtain an unknown humanADAMTS-1 gene from the human kidney cDNA library by plaquehybridization, using probes designed on the basis of the base sequenceof the known mouse ADAMTS-1 gene, but a desired gene was not obtained.One of the reasons of this failure was an insufficient homology of theprobes used. Further, the present inventors have carried out northernhybridization to analyze the mRNA of the human ADAMTS-1 protein, andconfirmed that the failure must also have occurred because mRNA isexpressed in a very small amount, and thus a very small number of thehuman ADAMTS-1 genes are copied in a cDNA library.

[0067] The gene of the present invention may be obtained by plaquehybridization, using probes designed on the basis of the base sequenceof the human ADAMTS-1 gene.

[0068] The resulting gene of the present invention may be expressed in,for example, a eucaryotic or procaryotic host to produce the protein ofthe present invention.

[0069] If a DNA fragment containing a desired gene is directlyintroduced into a host cell, the fragment is not reproduced. However, anextrachromosomal gene reproducible in a cell, such as a plasmid, may beused as a vector to prepare an expression plasmid. A vector which may beused preferably contains genetic information necessary for replicationin a host cell, can be independently replicated, is easily isolated andpurified from a host cell, and contains a detectable marker.

[0070] An expression vector containing the DNA of the present inventionmay be constructed in accordance with a host cell from variouscommercially available vectors. A method for introducing the DNA intothe vector is well known.

[0071] As a procaryotic host, there may be mentioned, for example, E.coli strains, such as XL1-Blue, HB101, JM109, DH5α, AG-1, K12 strain 294(ATCC 31446), B, χ1776 (ATCC 31537), C600, or W3110 (F-, λ-,prototrophic; ATCC 27375). Further, Bacillus strains, such as Bacillussubtibis, enteric bacteria, such as Salmonella typhimurium or Serratiamarcescens, or Pseudomonas strains may be used.

[0072] When the procaryotic host is used, an expression plasmid whichmay be used as a vector contains a promoter, an SD base sequence, and abase sequence necessary for initiating a protein synthesis, i.e., ATG,upstream of the gene of the present invention so as to express the gene.As a vector for E. coli strains, pUC19, pBR322, or pBR327 are generallyand widely used.

[0073] As a promoter, for example, triptophan promoter, P_(L) promoter,lac promoter, tac promoter, trc promoter, lpp promoter, or β-lactamasepromoter may be used. Examples of the marker gene are an ampicillinresistance gene or tetracycline resistance gene.

[0074] As a eucaryotic host, a yeast is generally and widely used. Ofthe yeasts, a Saccharomyces yeast can be advantageously used. As anexpression vector for the eucaryotic host such as a yeast, for example,YRp7 may be used

[0075] As a promoter of the expression vector for a yeast expression,for example, alcohol dehydrogenase (ADH), GAL10, 3-phosphoglyceratekinase, enolase, glyceraldehyde-3-phosphate dehydrogenase, or hexokinasemay be used. An example of a marker gene is a trpl gene.

[0076] As a replication origin, a stop codon, or other DNA sequencesused to control a transcription or translation in a yeast cell, usualknown DNA sequences suitable to the yeast cell may be used.

[0077] As a culture cell host of a higher animal, there may bementioned, for example, a rhesus renal cell, a mosquito larva cell, anAfrican Green Monkey kidney cell (COS-7 or COS-1), a murine fetalfibroblast, a Chinese hamster ovary cell or a dihydrofolatereductase-defective strain thereof, a human cervical epitheliocyte, ahuman fetal kidney cell, a moth ovary cell, a human myeloma cell, or amurine fibroblast cell.

[0078] The vector generally contains functional sequences to express theDNA of the present invention in a host cell, for example, a replicationorigin, a promoter, which should be upstream of the DNA of the presentinvention, a ribosome binding site, a polyadenylated site, and/or atranscription termination sequence. A preferable promoter is, forexample, an adenovirus 2 main late promoter, SV40 early promoter, SV40late promoter, or a promoter from cytomegalovirus, Rous sarcoma virus,or an eucaryote gene, such as estrogen inducible chick egg albumin gene,interferon gene, glucocorticoid inducible throsine aminotransferasegene, thymidine kinase gene, main early and late adenovirus gene,phosphoglycerate kinase gene, or a factor gene.

[0079] A replication origin from adenovirus, SV40, bovine papillomavirus (BPV), vesicular stomatitis virus (VSV), or a derivative vectorthereof may be used as the replication origin. In these cases, forexample, a neomycin resistance gene, a methotrexate resistantdihydrofolate reductase (DHR) gene, or a blasticidin S resistance genemay be used as the marker gene.

[0080] As an insect cell host, for example, BmN4 cell, Sf9 cell, Sf21cell, or an ovary cell of Trichoplusiani may be used. Further, a larvalsilkworm individual may be used as a host. A gene transfer to the insectcell can be carried out by co-infecting the insect cell with a virus DNAand a transfer vector containing a desired gene to be incorporated. Asthe virus DNA, for example, a Bombyx mori nuclear polyhedrosis virus, orAutographica californica multiple nuclear polyhedrosis virus may beused. As the transfer vector containing the desired gene to be inserted,for example, a polyhedrin promoter or p10 promoter vector may be used.The desired gene can be inserted downstream of the promoters. Thetransfer vector can be replicated in E. coli, but cannot be replicatedin an insect cell. Therefore, it is preferable to replicate many vectorsin E. coli, and then express them in the insect cell. According to thisprocess, a larger amount of expressed substances can be recovered incomparison with an animal cell.

[0081] The resulting expression plasmid may be transfected into anappropriate host cell, for example, a microorganism cell, such as E.coli yeast, or an animal cell, to produce the transformant of thepresent invention. The method for transfecting the DNA may be, forexample, a method of utilizing a competent cell treated with calciumchloride, a protoplast method, a calcium phosphate transfection method,or an electroporation method.

[0082] The pharmaceutical composition of the present invention comprises(1) the protein containing the amino acid sequence of SEQ ID NO: 1,particularly the human ADAMTS-1 protein, (2) the human ADAMTS-1 proteinvariation, or (3) the ADAMTS protein, as an active ingredient. In thepharmaceutical composition of the present invention, the activeingredient may be the mouse ADAMTS-1 protein. The proteins which may beused as the active ingredient of the pharmaceutical composition in thepresent invention have activities to influence hematopoietic functions,for example, activities to decrease the number of leukocytes andplatelets, and at the same time, increase the number of erythrocytes,when administered into a blood vessel.

[0083] It is possible to orally or parenterally administer thepharmaceutical composition according to the present invention, i.e., theprotein containing the amino acid sequence of SEQ ID NO: 1, particularlythe human ADAMTS-1 protein, the human ADAMTS-1 protein variation, or theADAMTS protein, alone or preferably together with a pharmaceutically orveterinarily acceptable ordinary carrier, to an animal, preferably amammal, particularly humans. The formulation is not particularly limitedto, but may be, for example, oral medicines, such as powders, finesubtilaes, granules, tablets, capsules, suspensions, emulsions, syrups,extracts or pills, or parenteral medicines, such as injections, liquidsfor external use, ointments, suppositories, creams for topicalapplication, or eye lotions.

[0084] The oral medicines may be prepared by an ordinary method using,for example, fillers, binders, disintegrating agents, surfactants,lubricants, flowability-enhancers, diluting agents, preservatives,coloring agents, perfumes, tasting agents, stabilizers, humectants,antiseptics, antioxidants, such as gelatin, sodium alginate, starch,corn starch, saccharose, lactose, glucose, mannitol,carboxylmethylcellulose, dextrin, polyvinyl pyrrolidone, crystallinecellulose, soybean lecithin, sucrose, fatty acid esters (such asglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acidester, or propyleneglycol fatty acid ester), talc, magnesium stearate,polyethylene glycol, magnesium silicate, silicic anhydride, or syntheticaluminum silicate.

[0085] For the parenteral administration, for example, an injection suchas a subcutaneous or intravenous injection, or the per rectumadministration may be used. Of the parenteral formulations, an injectionis preferably used.

[0086] When the injections are prepared, for example, water-solublesolvents, such as physiological saline or Ringer's solution,water-insoluble solvents, such as plant oil or fatty acid ester, agentsfor rendering isotonic, such as glucose or sodium chloride, solubilizingagents, stabilizing agents, antiseptics, suspending agents, oremulsifying agents may be optionally used, in addition to the proteincontaining the amino acid sequence of SEQ ID NO: 1, particularly thehuman ADAMTS-1 protein, the human ADAMTS-1 protein variation, or theADAMTS protein.

[0087] The pharmaceutical composition may be administered in the form ofa sustained release preparation using sustained release polymers. Forexample, the pharmaceutical composition of the present invention may beincorporated to a pellet made of ethylenevinyl acetate polymers, and thepellet may be surgically implanted in a tissue to be treated.

[0088] The pharmaceutical composition of the present invention maycontain the protein containing the amino acid sequence of SEQ ID NO: 1,particularly the human ADAMTS-1 protein, the human ADAMTS-1 proteinvariation, or the ADAMTS protein in an amount, but not particularlylimited to, of 0.0001 to 99% by weight, preferably 0.01 to 80% byweight, more preferably 0.01 to 50% by weight.

[0089] When the pharmaceutical composition of the present invention isutilized, the dose is not particularly limited, but varies with the kindof disease, the age, sex, body weight, or symptoms of the subject, amethod of administration, or the like. However, the protein containingthe amino acid sequence of SEQ ID NO: 1, particularly the human ADAMTS-1protein, the human ADAMTS-1 protein variation, or the ADAMTS protein maybe orally or parenterally administered at a dosage of about 0.0001 μg/kgto 10,000 μg/kg, preferably 0.001 μg/kg to 1,000 μg/kg, more preferably0.01 μg/kg to 100 μg/kg a day for an adult, usually once or divided intoup to four dosages.

[0090] The pharmaceutical composition of the present invention may beused not only for the pharmaceutical application but also for variousapplications. That is, the protein containing the amino acid sequence ofSEQ ID NO: 1, particularly the human ADAMTS-1 protein, the humanADAMTS-1 protein variation, or the ADAMTS protein may be administered inthe form of functional food or health food, together with a conventionalfood additive, or directly added to food as a food additive.

[0091] The pharmaceutical composition of the present invention containsthe protein containing the amino acid sequence of SEQ ID NO: 1,particularly the human ADAMTS-1 protein, the human ADAMTS-1 proteinvariation, or the ADAMTS protein, and thus is useful as, for example, anagent for reducing leukocytes, an agent for reducing platelets, or anagent for increasing erythrocytes.

[0092] It is generally known that, when inflammation occurs, leukocytesare activated in blood, move to an inflamed site, evolute and/or causepathosis. Therefore, it is believed that the human ADAMTS-1 protein ofthe present invention would be effective in the treatment of variousinflammatory diseases, such as rheumatic arthritis, psoriasis, asthma,hepatitis, Kawasaki disease, gout, adult respiratory distress syndrome(ARDS), Crohn's disease, ulcerative colitis, sepsis, or nephritis.Further, the human ADAMTS-1 protein of the present invention exhibits afunction to decrease the number of leukocytes and platelets, and thuswould be effective in a treatment of true hypervolemia. The humanADAMTS-1 protein of the present invention exhibits a function todecrease the number of platelets. Therefore, it is believed that thehuman ADAMTS-1 protein of the present invention would exhibit ananti-thrombotic action, and would be effective in the treatment ofcardiac infarction, cerebral inferction, or multi-organ failure. Thehuman ADAMTS-1 protein of the present invention exhibits a function tosignificantly increase the number of erythrocytes, and would beeffective in a treatment of anemia, as erythropoietin.

[0093] When lipopolysaccharide (LPS), an immunologically stimulatingsubstance, is administered to a mouse (for example, a 10 μg/mouse), anexpression of the mouse ADAMTS-1 gene is superinduced in a heart and akidney [J. Biol. Chem., 272, 556-562 (1997)]. Therefore, the mouseADAMTS-1 protein would possibly exhibit a protective function for aheart and a kidney, upon a lethally acute inflammation such as anendotoxin shock.

[0094] Thrombospondin (TSP) is known as a factor for inhibitingvascularization, i.e., specifically inhibiting a growth of endothelialcells [J. Cell. Biol., 111, 765-772 (1990)]. Further, it was reportedthat the proliferation and metastasis of cancer cells can be inhibitedby the induction of TSP in cancer cells [J. Cell. Biol., 111, 765-772(1990)]. Therefore, the human ADAMTS-1 protein would probably show afunction for inhibiting a cancer or metastasis. Further, a recent reporthad stated that TSP or disintegrin is involved in a bone formation[Biochem. Biophy. Res. Commun., 213, 1017-1025 (1995)]. Therefore, thehuman ADAMTS-1 protein could be applicable to the treatment of ametabolic bone disease, such as osteoporosis.

[0095] The immunologically reactive substance of the present inventionspecifically reacts the protein containing the amino acid sequence ofSEQ ID NO: 1, particularly the human ADAMTS-1 protein, the humanADAMTS-1 protein variation, or the ADAMTS protein. The immunologicallyreactive substance of the present invention includes, for example, anantibody (a monoclonal antibody or a polyclonal antibody), fragments ofthe antibody, such as, Fab, Fab′, F(ab′)₂, or Fv, and an antiserum. Theimmunologically reactive substance of the present invention specificallyreacts the human ADAMTS-1 protein, and therefore, the immunologicallyreactive substance of the present invention specifically reacts thehuman ADAMTS-1 protein, and therefore, is useful for analyzing the humanADAMTS-1 protein by immunological ways.

[0096] The monoclonal antibody of the present invention may be preparedby a known method, for example, the following method.

[0097] A physiological salt solution containing an antigen is mixed withan equal volume of complete Freund's adjuvant or incomplete adjuvant, oran equivalent thereof, such as Hunter's TiterMax™ (Funakoshi; Cat. No.YT001-00, Tokyo, Japan), until emulsified. The resulting emulsion isadministered subcutaneously, intraperitoneally, intravenously,intramuscularly, or intradermally to a mammal, for example, a mouse,rat, rabbit, or hamster, more particularly, a mouse, such as a BALB/cmouse, selected in view of a congeniality to a conventional myeloma cell(a first immunization). Then, the same procedure is repeated atintervals of two to four weeks for several immunizations, and finalimmunization is carried out using only the antigen solution. The spleensare removed aseptically several days after the final immunization, andspleen cells are prepared.

[0098] The resulting spleen cells are used for a cell fusion. The otherparent cells used for the cell fusion, that is, the myeloma cells, maybe known cell lines, such as, P3X63-Ag8(X63) [Nature, 256, 495-497(1975)], P3X63-Ag8U1 (P3U1) [Current Topics in Microbiology andImmunology, 81, 1-7 (1978)], or P3X63Ag8.653 (ATCC deposition No.CRL-1580).

[0099] The cell fusion may be carried out by ususal methods, forexample, the method of Milstein et. al. [Methods in Enzymology, 73, 3-47(1981)]. The resulting hybridomas are administered to mammals (forexample, mice), and desired monoclonal antibodies are separated andpurified from ascites of the mammals. The separation and purificationmethod used may be a known method, such as a dialysis ion exchangechromatography using an ammonium sulfate; an affinity columnchromatography using a protein A or protein G binding polysaccharidescarrier or an anti-mouse immunoglobrin antibody binding polysaccharidescarrier; dialysis, or lyophilization.

[0100] The polyclonal antibody of the present invention may be preparedby a known method, as indicated below. That is, a physiological saltsolution containing an antigen is mixed with an equal volume of completeFreund's adjuvant or incomplete adjuvant, or an equivalent thereof, suchas Hunter's TiterMax™ (Funakoshi; Cat. No. YT001-00, Tokyo, Japan),until emulsified. The resulting emulsion is administered subcutaneously,intraperitoneally, or intramuscularly to a mammal, for example, arabbit, or goat (a first immunization). Then, the same procedure isrepeated at intervals of two to four weeks for several immunizations.One or two weeks after a final immunization, blood is taken from acarotid artery or a heart of the mammal, and salted-out with ammoniumsulfate to prepare a serum.

[0101] The antibody fragment of the present invention may be prepared,for example, by digesting the polyclonal antibody or monoclonal antibodyof the present invention with a known protease by a conventional method,and then isolating and purifying by a conventional method.

[0102] Production of the human ADAMTS-1 protein is facilitated by animmunologically stimulating substance, such as LPS, and therefore, thehuman ADAMTS-1 protein may be used as a diagnostic marker of animmunological state in a method for extracorporeally detecting theimmunological state. More particularly, the extracorporeally detectingmethod of the present invention may be applied to a sample taken from asubject to be examined, to thereby detect an immunological state of animmune function of the subject, when the immune function is affected byvarious diseases, such as inflammation, cancer, cachexia, such as cancercachexia or infectious disease-related cachexia, infectious disease, orleukemia. If the immune function of the subject is normal, animmunological state corresponding to the immune function can bedetected.

[0103] The sample which may used in the present invention is notparticularly limited, so long as it has a possibility of including thehuman ADAMTS-1 protein. The sample may be a biological sample taken froma human, particularly a patient, for example, a humor, such as a tissue(e.g., cells) or an extract therefrom, blood, such as serum, and plasma,urine, or cerebrospinal fluid. A sample used in a conventional clinicalexamination may be used in the present invention without limitation.

[0104] In the analysis step of the human ADAMTS-1 protein in the sample,the sample is first brought into contact with the substanceimmunologically reactive to the human ADAMTS-1 protein. If the sampledoes not contain the human ADAMTS-1 protein, a reaction with theimmunologically reactive substance does not occur. If the samplecontains the human ADAMTS-1 protein, the immunologically reactivesubstance binds the human ADAMTS-1 protein, and a complex of theimmunologically reactive substance and the human ADAMTS-1 protein isformed in an amount correlated with that of the human ADAMTS-1 proteinpresent in the sample. The complex may be easily detected by a knownmethod, and therefore, an existence of the human ADAMTS-1 protein in thesample can be detected by detecting the existence of the complex, or anamount of the human ADAMTS-1 protein in the sample can be measured bymeasuring the amount of the complex. The human ADAMTS-1 protein in atissue or a cell may be measured, using a tissue section sample or acell sample in a fluorescent antibody technique or an enzyme antibodytechnique.

[0105] The immunologically reactive substance capable of immunologicallyreacting the human ADAMTS-1 protein includes an anti-human ADAMTS-1protein antiserum, an anti-human ADAMTS-1 protein polyclonal antibody,of an anti-human ADAMTS-1 protein monoclonal antibody, or a fragmentthereof. The immunologically reactive substance may be used singly or incombination thereof. The fragment includes, for example, Fab, Fab′,F(ab′)₂, or Fv.

[0106] In the method for immunologically analyzing the human ADAMTS-1protein according to the present invention, the sample is brought intocontact with the immunologically reactive substance capable ofimmunologically reacting the human ADAMTS-1 protein, and a complex ofthe human ADAMTS-1 protein and the immunologically reactive substance isformed. Then, the human ADAMTS-1 protein bound to the antibody isdetected and the amount thereof is measured by an immunochemical method,to thereby find a level of the human ADAMTS-1 protein in the sample.

[0107] The immunochemical method principally may be, for example, anyconventional immunoassay, for example, EIA, ELISA, RIA or the like. Theimmunochemical methods are generally classified as follows:

[0108] (1) Competitive Assay:

[0109] A sample containing an unknown amount of antigens and a givenamount of labeled antigens is competitively reacted with a given amountof antibodies, and then an activity of the labeled antigens bound to theantibodies or an activity of the labeled antigens not bound to theantibodies is measured.

[0110] (2) Sandwich Assay:

[0111] An excess amount of antibodies immobilized on carriers is addedand reacted to a sample containing an unknown amount of antigens (afirst reaction). Then, a given excess amount of labeled antibodies isadded and reacted therewith (a second reaction). An activity of thelabeled antibodies on the carriers is measured. Alternatively, anactivity of the labeled antibodies which are not on the carriers ismeasured. The first reaction and the second reaction may be carried outat the same time, or sequentially.

[0112] When a labeling agent is a radioactive isotope, a well counter ora scintillation counter may be used for measurement. When the labelingagent is an enzyme, an enzymatic activity can be measured by colorimetryor fluorimetry, after adding a substrate and allowing to stand. When thelabeling agent is a fluorescent substance or an luminescent substance, aknown method therefor may be used, respectively.

[0113] In addition to the above methods, recently a western blottingmethod has been used wherein electrophoresed proteins are transferredonto a filter such as a nitrocellulose membrane, and a target protein isdetected with an antibody. The western blotting method may also be usedin the detection of the human ADAMTS-1 protein in the present invention.

[0114] The antibody used in the above methods can be labeled with anappropriate marker. Examples are a radioactive isotope, an enzyme, afluorescent substance, or a luminescent substance, by a known method oflabeling antibodies.

[0115] The radioactive isotope may be, for example, ¹²⁵I, ¹³¹I, ³H, ⁴C,or ³⁵S

[0116] Preferably, the enzyme used is stable and has a large specificactivity. Examples of the enzyme are a glycosidase (such as,β-galactosidase, β-glucosidase, β-glucuronidase, β-fructosidase,α-galactosidase, α-glucosidase, or α-mannosidase), an amylase (such as,α-amylase, β-amylase, isoamylase, glucoamylase, or taka-amylase), acellulase, or a carbohydrase such as lysozyme; a urease, or an amidasesuch as asparaginase; a choline esterase, such as acetylcholinesterase,a phosphatase, such as alkaline phosphatase, a sulfatase, an esterasesuch as lipase; a nuclease such as deoxyribonuclease or ribonuclease; aniron porphyrin enzyme, such as a catalase, peroxidase or cytochromeoxidase; a copper enzyme, such as a tyrosinase or ascorbate oxidase;dehydrogenase, such as an alcohol dehydrogenase, malate dehydrogenase,lactate dehydrogenase, or isocitrate dehydrogenase.

[0117] The fluorescent substance may be, for example, fluorescamine, ora fluorescence isothiocyanate, and the luminescent substance may be, forexample, luminol, a luminol derivative, luciferin or lucigenin. A signalfrom the above label may be detected by known methods.

[0118] The labeling agent can be bound to antibodies by any conventionalmethod, such as a chloramin T method [Nature, 194, 495-496, (1962)], aperiodic acid method [Journal of Histochemistry and Cytochemistry, 22,1084-1091, (1974)], or a maleimide method [Journal of Biochemistry, 79,233-236, (1976)].

[0119] An EIA method as one of the above measurement methods will bementioned hereinafter. A sample is added to the first anti-humanADAMTS-1 protein antibodies immobilized on a carrier (such as an assayplate), and the anti-human ADAMTS-1 protein antibodies are bound to thehuman ADAMTS-1 proteins to form complexes. To the complexes, the secondanti-human ADAMTS-1 protein antibodies labeled with enzyme (such asperoxidase) are added to react with the complexes to form “firstantibody/human ADAMTS-1 protein/second antibody” complexes. To theresulting “first antibody/human ADAMTS-l protein/second antibody”complexes, a substrate for the enzyme label (such as peroxidase) isadded, and an absorbance or fluorescent strength of products of theenzymatic reaction is measured, whereby enzymatic activities of theenzyme labels attached to the “first antibody/human ADAMTS-1protein/second antibody” complexes are measured. A series of the aboveprocedures is carried out in advance for a standard solution containinga known amount of the human ADAMTS-1 protein, and a standard curve basedon the relationship between the human ADAMTS-1 protein and theabsorbance or fluorescent strength is prepared. A comparison is madebetween the standard curve and absorbance or fluorescent strength for asample containing an unknown amount of the human ADAMTS-1 proteins, andthe amount of the human ADAMTS-1 proteins in the sample can be measured.

[0120] Another EIA method will be mentioned hereinafter. A sample isbrought into contact with a carrier (such as an assay plate) toimmobilize the human ADAMTS-1 proteins in the sample onto the carrier.Then, the anti-human ADAMTS-1 protein antibodies (first antibodies) areadded thereto to form complexes of the human ADAMTS-1 protein and thefirst antibody. To the complexes are added anti-first antibodyantibodies (second antibodies) labeled with enzyme (such as peroxidase),to react with the complexes to form “human ADAMTS-1 protein/firstantibody/second antibody” complexes. To the resulting “human ADAMTS-1protein/first antibody/second antibody” complexes is added a substratefor the enzyme label (such as peroxidase), and the absorbance orfluorescent strength of products of the enzymatic reaction is measured,whereby enzymatic activities of the enzyme labels attached to the “humanADAMTS-1 protein/first antibody/second antibody” complexes are measured.A series of the above procedures is carried out in advance for astandard solution containing a known amount of the human ADAMTS-1protein, and a standard curve based on the relationship between thehuman ADAMTS-1 protein and the absorbance or fluorescent strength isprepared. A comparison is made between the standard curve and theabsorbance or fluorescent strength for a sample containing an unknownamount of the human ADAMTS-1 proteins, and the amount of the humanADAMTS-1 proteins in the sample can be measured.

[0121] Further, an RIA method will be mentioned hereinafter. A sample isadded to the first anti-human ADAMTS-1 protein antibodies immobilized ona carrier (such as a test tube), and the anti-human ADAMTS-1 proteinantibodies are bound to the human ADAMTS-1 proteins to form complexes.To the complexes are added the second anti-human ADAMTS-1 proteinantibodies labeled with radioactive isotope (such as ¹²⁵I), to reactwith the complexes to form “first antibody/human ADAMTS-1 protein/secondantibody” complexes. A radioactivity of the resulting “firstantibody/human ADAMTS-1 protein/second antibody” complexes is measured.A series of the above procedures is carried out in advance for astandard solution containing a known amount of the human ADAMTS-1protein, and a standard curve based on the relationship between thehuman ADAMTS-1 protein and the radioactivity is prepared. A comparisonis made between the standard curve and the radioactivity for a samplecontaining an unknown amount of the human ADAMTS-1 proteins, and theamount of the human ADAMTS-1 proteins in the sample can be measured.

[0122] Another RIA method will be mentioned hereinafter. A sample isbrought into contact with a carrier (such as a test tube) to immobilizethe human ADAMTS-1 proteins in the sample onto the carrier. Then, theanti-human ADAMTS-1 protein antibodies (first antibodies) are addedthereto to form complexes of the human ADAMTS-1 protein and the firstantibody. To the complexes are added anti-first antibody antibodies(second antibodies) labeled with radioactive isotope (such as ¹²⁵), toreact with the complexes to form “human ADAMTS-1 protein/firstantibody/second antibody” complexes. A radioactivity of the resulting“human ADAMTS-1 protein/first antibody/second antibody” complexes ismeasured. A series of the above procedures is carried out in advance fora standard solution containing a known amount of the human ADAMTS-1protein, and a standard curve based on the relationship between thehuman ADAMTS-1 protein and the radioactivity is prepared. A comparisonis made between the standard curve and the radioactivity for a samplecontaining an unknown amount of the human ADAMTS-1 proteins, and theamount of the human ADAMTS-1 proteins in the sample can be measured.

[0123] In the method for analyzing the mRNA of the human ADAMTS-1protein in a sample, the sample is reacted with a polynucleotidecontaining a base sequence complementary to that of the mRNA of thehuman ADAMTS-1 protein, and the resulting complex of the mRNA of thehuman ADAMTS-1 protein and the polynucleotide is detected, or the amountof the complex is measured to thereby analyze the mRNA of the humanADAMTS-1 protein.

[0124] The polynucleotide contains a sequence complementary orsubstantially complementary to that of a part of the mRNA transcribedfrom a selected gene (DNA), and thus forms a double strand with the mRNAtranscribed from the target gene. It is believed that any polynucleotidesufficiently complementary to form a stable complex with a target mRNAcan be used. The polynucleotide able to be used in the present inventionmay be complementary to substantially any region in a target mRNA. Thepolynucleotide can be used as a DNA probe for detecting an increase or adecrease of expression of the mRNA specific to the human ADAMTS-1protein gene. That is, the polynucleotide is specifically attached tothe mRNA of the target human ADAMTS-1 protein, and forms a molecularhybrid, whereby a degree of expression of the human ADAMTS-1 protein incells can be detected.

[0125] The polynucleotide able to be used in the present invention maybe prepared by appropriately selecting a base sequence complementary toa specific base sequence of the mRNA of the target human ADAMTS-1protein, and using a known DNA synthesizer, a known PCR apparatus, agene cloning or the like. Various length polynucleotides may be used,but the polynucleotide preferably has 10 or more bases, more preferably17 or more bases.

[0126] The polynucleotide may be a non-modified polynucleotide or apolynucleotide analogue. An appropriate analogue may be, for example, anethyl or methyl phosphate analogue, or a phosphorothioatedpolydeoxynucleotide [Nucleic Acids Res., 14, 9081-9093, (1986); J. Am.Chem. Soc., 106, 6077-6079, (1984)], with recent improvement in theproduction of polynucleotide analogue, for example, a2′-O-methylribonucleotide [Nucleic Acids Res., 15, 6131-6148, (1987)],or a conjugated RNA-DNA analogue, i.e., chimera polynucleotide [FEBSLett., 215, 327-330, (1987)], may be used.

[0127] The selected polynucleotide may be of any kind, for example, mayhave an electrical charge or no electrical charge. The polynucleotidemay be labeled with a known labeling agent, such as a radioactiveisotope, or a fluorescent substance by a conventional method, so as tocarry out the above experiment in vitro or in vivo. The radioactiveisotope may be, for example, ¹²⁵I, ¹³¹I, ³H, ¹⁴C, ³²P, or ³¹S. Of theseradioactive isotopes, it is preferable to label the polynucleotide with³²P by a random primer method [Anal. Biochem., 132, 6-13, (1983)].Further, a fluorescent coloring agent forming a derivative may be usedas a labeling agent, as this enables an easy handling with a low riskfactor. As the fluorescent coloring agent, any coloring agents capableof binding the polynucleotide may be used. For example, fluorescein,rhodamin, Texas red, 4-fluoro-7-nitrobenzofurazane (NBD), coumarin,fluorescamine, succinyl fluorescein, or dansyl may be preferably used.

[0128] An amount of an mRNA of the human ADAMTS-1 protein may bemeasured by a northern blotting method, using cDNA of the human ADAMTS-1protein as follows: an mRNA is extracted and isolated from any somaticcell or tissue, then the isolated mRNA is electrophoresed on an agarosegel and transferred onto a nitro cellulose or nylon membrane, and thenreacted with a labeled human ADAMTS-1 protein cDNA probe to measure anamount of the mRNA of the human ADAMTS-1 protein. The human ADAMTS-1protein cDNA probe used is a DNA complementary to the human ADAMTS-1protein mRNA, and has preferably 17 or more bases.

[0129] The agent for analyzing an immunological state according to thepresent invention contains, as a main ingredient, the immunologicallyreactive substance capable of immunologically reacting the humanADAMTS-1 protein. The immunologically reactive substance capable ofimmunologically reacting the human ADAMTS-1 protein may be, for example,an anti-human ADAMTS-1 protein antiserum, an anti-human ADAMTS-1 proteinpolyclonal antibody, or an anti-human ADAMTS-1 protein monoclonalantibody, of a fragment of the antibodies.

[0130] The agent for analyzing an immunological state according to thepresent invention may contain, as a main ingredient, the polynucleotidecontaining the base sequence complementary to that of an mRNA of thehuman ADAMTS-1 protein, instead of the immunologically reactivesubstance.

[0131] The human ADAMTS-1 protein per se or an mRNA of the humanADAMTS-1 protein in a sample can be analyzed according to the abovemethods using the agent for analyzing an immunological state accordingto the present invention, and the immunological state of a subject to beexamined, wherein the immune function is affected by various diseases,can be judged from the result.

EXAMPLES

[0132] The present invention now will be further illustrated by, but isby no means limited to, the following Examples.

Example 1

[0133] Isolation of the Human ADAMTS-1 cDNA and Determination of theBase Sequence Thereof

[0134] As primers for a PCR method, a DNA [hereinafter referred to as a“forward primer (1)”] having the base sequence (i.e., the base sequenceof SEQ ID NO: 4: AGAACCTGTG GTGGTGGAGT TCAATACACA) corresponding to anamino acid sequence (i.e., the amino acid sequence of SEQ ID NO: 3: ArgThr Cys Gly Gly Gly Val Gln Tyr Thr) contained in the firstthrombospondin (TSP) domain from the N-terminus among three TSP domainsof the mouse ADAMTS-1 protein [J. Biol. Chem., 272, 556-562 (1997)], anda DNA [hereinafter referred to as a “back primer (1)”] having the basesequence (i.e., the base sequence of SEQ ID NO: 5: CCTCTTAACT GCACTGTGTCAGTGTGCAAA AG) complementary to the base sequence encoding amino acidsin the C-terminus of the mouse ADAMTS-1 protein and the base sequence inthe vicinal regions (i.e., the regions upstream and downstream of theC-terminus) were chemically synthesized.

[0135] To 99 μl of a solution containing 0.5 μM forward primer (1), 0.5μM back primer (1), 0.5 units of Taq polymerase (Ex Taq polymerase;Takarashuzo, Kyoto, Japan), and 40 μM 4dNTP in a PCR buffer [10 mMTris-HCl (pH 8.3), 50 mM KCl] (Ex Taq buffer; Takarashuzo, Kyoto,Japan), 1 μl of a human kidney cDNA library (Marathon-Ready cDNA;Clontech Lab. Inc., Palo Alto, Calif., USA) was added as a template DNA.A PCR method was performed at an annealing temperature lower than thatused in a standard PCR method. That is, the PCR reaction was carried outby repeating a cycle composed of denaturation at 94° C. for 30 seconds,annealing at 50° C. for 30 seconds, and DNA synthesis at 72° C. for 2minutes, 40 times, i.e., 40 cycles.

[0136] A sample (5 μl) was taken from the resulting reaction mixture,and electrophoresed on 1% agarose gel. As shown in FIG. 1, a single DNAband of 1.2 Kb was observed. The rest of the reaction mixture waselectrophoresed, and the DNA fragment of 1.2 Kb (hereinafter sometimesreferred to as a “Flag. 1 DNA fragment”) was recovered from alow-melting-point agarose gel. Then, the Flag. 1 DNA fragment was clonedin a pCR™ 2.1 vector (Invitrogen Corp., San Diego, Calif., USA).

[0137] A part (303 bp) of the base sequence of the cloned Flag. 1 DNAfragment was determined by an automatic DNA sequencer (DSQ1000; ShimadzuCorp., Kyoto, Japan). A homology search between the partial basesequence of the cloned Flag. 1 DNA fragment and the base sequence of themouse ADAMTS-1 was conducted to find that a homology in the basesequences was 77.4%. The partial base sequence of the Flag. 1 DNAfragment and a partial base sequence of the mouse ADAMTS-1 gene having ahomology therewith are shown in FIG. 2. The symbol “:” in FIG. 2 meansthat a base in the Flag. 1 DNA fragment is identical to a correspondingbase in the mouse ADAMTS-1 gene.

[0138] Further, a dot hybridization [Biochemistry, 16, 4743-4749 (1977)]was conducted to find that a mouse ADAMTS-1 cDNA labeled with ³²P by arandom primed DNA-labeling kit (Boehringer Manmheim GmbH, Germany) washybridized with the Flag. 1 DNA fragment. The results are shown in FIG.3. A pCR™2.1 vector as a control was not hybridized with ³²P-labeledmouse ADAMTS-1 cDNA, whereas the mouse ADAMTS-1 cDNA and the Flag. 1 DNAfragment were hybridized with ³²P-labeled mouse ADAMTS-1 cDNA.

[0139] The results of the homology search and dot hybridization meanthat the Flag. 1 DNA fragment is a part of the human ADAMTS-1.

[0140] To obtain a DNA fragment upstream of the Flag. 1 DNA fragment, aRapid amplification of cDNA ends (RACE) method using a Marathon cDNAAmplification kit (Clontech Lab. Inc., Palo Alto, Calif., USA) wasperformed as follows. That is, as back primers for the base sequence ofthe Flag. 1 DNA fragment, a DNA primer (hereinafter sometimes referredto as a “GSP-1 primer”) having the base sequence (i.e., the basesequence of SEQ ID NO: 6: CCTCTTAACT GCACTGTGTC AGT) complementary tothe base sequence of the 3′-end region of the Flag. 1 DNA fragment, anda DNA primer (hereinafter sometimes referred to as a “GSP-2 primer”)having the base sequence (i.e., the base sequence of SEQ ID NO: 7:CAGGCCCACT CCCAAAGGAA GCTT) complementary to the base sequence of the5′-end region of the Flag. 1 DNA fragment were chemically synthesized.As forward primers, an AP1 primer and an AP2 primer attached to theabove kit were used. The AP1 primer had the base sequence of SEQ ID NO:8:

[0141] CCATCCTAAT ACGACTCACT ATAGGGC,

[0142] and the AP2 primer had the base sequence of SEQ ID NO: 9:

[0143] ACTCACTATA GGGCTCGAGC GGC.

[0144] After 5 μl of a human kidney cDNA library (Marathon-Ready cDNA;Clontech Lab. Inc., Palo Alto, Calif., USA), 1 μl of the AP1 primer, 1μl of 10 μM GSP-1 primer, 1 μl of a Taq polymerase (0.5 unit; Ex Taqpolymerase), 1 μl of an anti-Taq polymerase antibody (Taq StartAntibody; Clontech Lab. Inc., Palo Alto, Calif., USA), 5 μl of a PCRbuffer having a 10-fold concentration [100 mM Tris-HCl (pH 8.3), 500 mMKCl] (Clontech Lab. Inc., Palo Alto, Calif., USA), and 36 μl ofdistilled water were mixed, a PCR reaction was performed. The PCRreaction was carried out by repeating a cycle composed of a step at 94°C. for 30 seconds and a step at 68° C. for 4 minutes, 35 times, i.e., 35cycles. The resulting reaction mixture was diluted to 50-fold with 10 mMTricine-EDTA buffer (Clontech Lab. Inc., Palo Alto, Calif., USA).

[0145] Then, 5 μl of the diluted liquid, 1 μl of the AP2 primer, 1 μl ofa 10 μM GSP-2 primer, 1 μl of a Taq polymerase (0.5 unit; Ex Taqpolymerase), 1 μl of an anti-Taq polymerase antibody (Taq StartAntibody), 5 μl of a PCR buffer having a 10-fold concentration (ClontechLab. Inc., Palo Alto, Calif., USA), and 36 μl of distilled water weremixed, and a PCR reaction was then performed. The PCR reaction wascarried out by repeating a cycle composed of a step at 94° C. for 30seconds and a step at 68° C. for 4 minutes 20 times, i.e., 20 cycles.

[0146] A sample (5 μl) was taken from the resulting reaction mixture,and electrophoresed on 1% agarose gel. As shown in FIG. 4, a single DNAband of about 1.2 Kb was observed. The DNA fragment (hereinaftersometimes referred to as a “Flag. 2 DNA fragment”) was cloned in apCR™2.1 vector by a conventional method, and the full-length basesequence of the Flag. 2 DNA fragment determined by a Dye TerminatorCycle Sequencing method (Perkin Elmer Japan, Urayasu, Japan).

[0147] Further, the full-length base sequence of the Flag. 1 DNAfragment was determined by the Dye Terminator Cycle Sequencing method(Perkin Elmer Japan, Urayasu, Japan). From the obtained base sequencesof the Flag. 1 DNA fragment and the Flag. 2 DNA fragment, thefull-length base sequence of the human ADAMTS-1 cDNA was determined. Thefull-length base sequence (2184 bp including a stop codon) is shown inSEQ ID NO: 2 and the amino acid sequence (727 amino acid residues) ofthe human ADAMTS-1 protein deduced from the above base sequence is shownin SEQ ID NO: 1. The partial base sequence of the Flag. 1 DNA fragmentas shown in FIG. 2 contains some bases not identical to the full-lengthbase sequence of the human ADAMTS-1 cDNA (SEQ ID NO: 2). However, thepartial base sequence as shown in FIG. 2 was an interim sequenceobtained in the process of determining the base sequence. The basesequence of SEQ ID NO: 2 is a correct base sequence of the humanADAMTS-1 cDNA which has been finally determined.

[0148] The human ADAMTS-1 protein is cysteine-rich, and contains manybasic amino acids such as lysine and arginine in the C-terminal region,and two N-glycosylation sites (the 307th to 309th amino acids and 524thto 526th amino acids).

[0149] The homology between the base sequences of the human ADAMTS-1gene and the mouse ADAMTS-1 gene is shown in FIGS. 5 to 9, and thehomology in the amino acid sequences deduced from the base sequences isshown in FIGS. 10 to 12.

[0150] In FIGS. 5 to 9, the symbol “*” means that a base of the humanADAMTS-1 gene is identical to the corresponding base of the mouseADAMTS-1 gene.

[0151] In FIGS. 10 to 12, the symbol “*” means that an amino acidresidue of the human ADAMTS-1 protein is identical to the correspondingamino acid residue of the mouse ADAMTS-1 protein. In FIGS. 10 to 12,“MMP domain” means a matrix metalloproteinase domain; the line betweenthe 11th and 12th amino acids indicates the starting site of the matrixmetalloproteinase domain; “DI domain” means the disintegrin domain; theline between the 234th and 235th amino acids indicates the starting siteof the disintegrin domain; “TSP domain” means the thrombospondin domain;and amino acid sequences in boxes (three occurrences) mean thethrombospondin domain.

[0152] The homology in the base sequences between the human ADAMTS-1 andthe mouse ADAMTS-1 was 85.5% and that in the amino acid sequences was90.1%. The results show that ADAMTS-1 is the protein of which, betweenmouse and human, the sequences have been preserved.

Example 2

[0153] Preparation of the Human ADAMTS-1 Fusion Protein in E. coli

[0154] (1) Construction of an expression vector for E. coli

[0155] To introduce a SmaI site at the 5′-side and a NotI site at the3′-side into the DNA encoding a partial region downstream of the MMPdomain in the full-length human ADAMTS-1 protein, a forward primer (2)having the base sequence of SEQ ID NO: 10:

[0156] CACCCCGGGA GGAAGAAGCG ATTTGTGTCC AGCCCCCGTT ATG,

[0157] and a back primer (2) having the base sequence of SEQ ID NO: 11:

[0158] GTGGCGGCCG CCCTCTTAAC TGCACTGTGT CAGTGTGCAA AA were chemicallysynthesized.

[0159] After 5 μl of the forward primer (2), 5 μl of the back primer(2), 1 μl of a human kidney cDNA library (Marathon-Ready cDNA; ClontechLab. Inc., Palo Alto, Calif., USA), 1 μl of a Taq polymerase (0.5 unit;Ex Taq polymerase), 1 μl of an anti-Taq polymerase antibody (Taq StartAntibody; Clontech Lab. Inc., Palo Alto, Calif., USA), 10 μl of a PCRbuffer having a 10-fold concentration (Clontech Lab. Inc., Palo Alto,Calif., USA), 8 μl of 2.5 mM 4dNTP (Takarashuzo, Kyoto, Japan), and 69μl of distilled water were mixed, a PCR reaction was performed. The PCRreaction was carried out by repeating a cycle composed of a step at 94°C. for 1 minute, a step at 55° C. for 45 seconds, and a step at 72° C.for 2 seconds 40 times, i.e., 40 cycles.

[0160] A sample (5 μl) was taken from the resulting reaction mixture,and electrophoresed on 1% agarose gel. As shown in FIG. 13, a single DNAband of about 2.2 Kb was obtained. The DNA fragment of about 2.2 Kb wascloned in the pCR™ 2.1 vector by a conventional method, and a largequantity of the plasmids produced [Nucleic Acids Res., 9, 2989-2998(1981)]. The resulting plasmids prepared on a large scale were treatedwith restriction enzymes, SmaI (Takarashuzo, Kyoto, Japan) and NotI(Takarashuzo, Kyoto, Japan) to obtain a DNA fragment of about 2.2 Kb.The DNA fragment of about 2.2 Kb was cloned in a SmaI-NotI site of anexpression vector for E. coli pGEX-5X-1 [Infect Immun., 58, 3909-3913(1990)] (Pharmacia Biotech, Uppsala, Sweden). The resulting expressionplasmid was named pG/ADAMTS-1. The structure of the plasmid pG/ADAMTS-1is schematically shown in FIG. 14. It is believed that the ADAMTS-1protein is expressed in the form of a fusion protein (molecularweight=about 96 Kd) with a glutathione-S-transferase (hereinaftersometimes referred to as a “GST”) (molecular weight=about 26 Kd). InFIG. 14, “Ori” means a replication origin, “Amp^(R)” means an ampicillinresistance gene, and “laq I^(q)” means a laq repressor.

[0161] (2) Expression of the GST-human ADAMTS-1 fusion protein in E.coli

[0162] The plasmid pG/ADAMTS-1 was introduced into an E. coli BL-21strain having a low protease activity (Pharmacia Biotech, Uppsala,Sweden) by a conventional method [Proc. Natl. Acad. Sci. USA, 69,2110-2114 (1972)]. E. coli clones containing the plasmid were isolatedas ampicillin resistance strains. Five ampicillin resistance strains(hereinafter referred to as “clone #1” to “clone #5”) randomly selectedfrom the strains were used to inoculate 2 ml of 2×YT medium (prepared bydissolving 16 g of trypton, 10 g of yeast extract, and 5 g of NaCl in 1liter of distilled water; pH 7.2), and cultured overnight at 37° C.Then, a set of two test tubes containing 1800 μl of an LB culture mediumcontaining ampicillin (100 μg/ml) was prepared for each clone. To eachtest tube, 200 μl of the overnight culture was poured. Then afterincubating at 37° C. for 2 hours, 20 μl (final concentration=1.0 mM) ofisopropyl-β-D-thio-galactopyranoside (IPTG) (Takarashuzo, Tokyo, Japan),an expression-inducer, was added to one of the two test tubes.Thereafter, the test tubes were incubated at 37° C. for 2 hours. Theexpression-inducer was not added to the other test tube, which was usedas a control.

[0163] Microorganisms were harvested by centrifugation (14000 rpm, 1minute) from 1 ml of the culture, using a microcentrifuging apparatus,then suspended in 100 μl of a phosphate-buffered solution (140 mM NaCl,2.7 mM KCl, 10 mM Na₂ HPO₄, 1.8 mM KH₂ PO₄, pH 7.2; hereinafter referredto as a PBS) and thereafter dissolved in 100 μl of a 2× sample buffer(0.25 M Tris-HCl, 2% SDS, 3% glycerol, 10% β-mercaptoethanol, 0.01%bromophenol blue; pH 6.8). The resulting solution (10 μl) was subjectedto an SDS-polyacrylamide gel electrophoresis (hereinafter sometimesreferred to as an “SDS-PAGE”), and an expression induction of thedesired protein was confirmed by a Coomassie staining method.

[0164] The results are shown in FIG. 15 wherein “+” lanes show theresults of the electrophoresis of E coli incubated with the expressioninducer, IPTG, and “−” lanes show the results of the electrophoresis ofE coli incubated without the expression inducer, IPTG. In each “+” lane,a protein having a molecular weight of about 100 Kd was observed. Thisis identical to an expected molecular weight of the GST-human ADAMTS-1fusion protein, i.e., about 96 Kd. However, such a protein was notobserved in the “−” lanes.

[0165] of five clones (“clone #1” to “clone #5”), the clone #2 exhibitedthe highest expression, and thus was used in the following Examples.

[0166] (3) Extraction and Isolation of the GST-human ADAMTS-1 FusionProtein

[0167] After overnight incubation, 1 ml of the culture of the clone #2was added to 100 ml of a 2×YT culture medium containing 100 μg/mlampicillin, and incubated at 37° C. When an absorbance at 600 nm reachedabout 0.5, 1 ml of a 100 mM IPTG was added to the culture, and thenincubation was continued for 2 hours.

[0168]E. coli was harvested by centrifugation (3000 rpm, 30 minutes)from the culture, and then suspended in 8 ml of PBS. To the suspension,1 ml of 0.5 M EDTA solution and 1 ml of 25 mg/ml lysozyme solution wereadded, and the whole was allowed to stand in ice for 30 minutes. After110 μl of Triton X-100 was added, the microorganisms were disrupted onice by a sonicator (TAITEC, Koshigaya, Japan). The disruption liquid wascentrifuged (8000 rpm, 4° C., 10 minutes), then the resultingprecipitate suspended in 30 ml of PBS containing 1.0% Triton X-100, andthereafter centrifuged (8000 rpm, 4° C., 10 minutes).

[0169] The resulting precipitate was suspended in 2 ml of a 10 mM EDTAsolution, and then, 50 ml of a 50 mM Tris-HCl buffer (pH 8.5) containing8 M urea and 1% mercaptoethanol was added. After a thorough admixing,the mixture was centrifuged (15000 rpm, 4° C., 5 minutes). The resultingsupernatant was dialyzed against 5 liters of a 10 mM Tris-HCl buffer (pH8.5) at 4° C. The dialyzed solution was centrifuged (15000 rpm, 4° C., 5minutes), and the resulting supernatant then adsorbed to an anionchromatography (Econo-Pac High Q; Bio-Rad Lab., Hercules, Calif., USA).Fractions eluted with 0.2 to 0.4 M NaCl were dialyzed against 3 litersof PBS, and then adsorbed to 1 ml of glutathione Sepharose 4 B(Pharmacia Biotech, Uppsala, Sweden) [Nucleic Acids Res., 9, 2989-2998(1981)].

[0170] The glutathione Sepharose 4 B was washed with 50 ml of PBS, andthen eluted with 8 ml of 10 mM glutathione solution [Nucleic Acids Res.,9, 2989-2998 (1981)]. Fractions exhibiting a high GST activity detectedby a GST detecting kit (Pharmacia Biotech, Uppsala, Sweden) were pooled.

[0171] A part of the fractions was subjected to an SDS-PAGE, and aCoomassie staining was performed. As shown in FIG. 16, a GST-humanADAMTS-1 fusion protein was confirmed from the molecular weight. Thedesired protein (about 1 μg) was extracted and purified from 100 ml ofE. coli culture.

[0172] The resulting fusion protein contains a site which may be brokenwith a Factor Xa or the like, between the GST and the human ADAMTS-1protein, and therefore, the human ADAMTS-1 protein can be obtained bydigesting the fusion protein with the proteinase. The human ADAMTS-1protein may be used as an antigen to prepare an antibody.

Example 3

[0173] Examination of Activities of GST-human ADAMTS-1 Fusion Protein onInfluencing Hematopoietic Functions

[0174] To examine the activities of the GST-human ADAMTS-1 fusionprotein on influencing hematopoietic functions, a large-scalepreparation of the GST-human ADAMTS-1 fusion protein was carried out inaccordance with the process disclosed in Example 2 (3), and about 30 μgof the desired protein was obtained from 3 liters of E. coli culture.

[0175] The functions thought to influence the number of blood cells by asingle dosage of the GST-human ADAMTS-1 fusion protein to a tail vein ofa mouse were examined, as the activities influencing hematopoieticfunctions. The examining system can be conducted with a small amount ofa protein to be examined, and enables a quick elucidation of abiological activity. In a control test, a GST protein extracted andpurified by the process disclosed in Example 2(3) from E. colitransformed with a vector pGEX-5X-l was used.

[0176] The GST-human ADAMTS-1 fusion protein (1 μg) was administered toeight C₅₇BL/6N mice (Charlese river Japan, Yokohama, Japan) (male, 7weeks old) at a tail vein. The numbers of leukocytes, erythrocytes, andplatelets were counted 3 hours and 24 hours after the administration. Inthe control test, the GST protein (1 μg) was administered to eightC₅₇BL/6N mice (Charlese river Japan, Yokohama, Japan) (male, 7 weeksold) at a tail vein. The numbers of leukocytes, erythrocytes, andplatelets were counted 3 hours and 24 hours after the administration.

[0177] The results are shown in FIG. 17. It is apparent from FIG. 17that the number of leukocytes and platelets is significantly decreased,and the number of erythrocytes is significantly increased in the mice towhich the GST-human ADAMTS-1 fusion protein was administered, incomparison with the control tests.

[0178] Industrial Applicability

[0179] According to the protein of the present invention, hematopoieticfunctions can be controlled, for example, the number of leukocytes andplatelets can be decreased, and at the same time, the number oferythrocytes can be increased.

[0180] As above, the present invention is explained with reference toparticular embodiments, but modifications and improvements obvious tothose skilled in the art are included in the scope of the presentinvention.

1 14 1 727 PRT Homo sapiens 1 Met Asp Ile Cys Arg Ile Arg Leu Arg LysLys Arg Phe Val Ser Ser 1 5 10 15 Pro Arg Tyr Val Glu Thr Met Leu ValAla Asp Gln Ser Met Ala Glu 20 25 30 Phe His Gly Ser Gly Leu Lys His TyrLeu Leu Thr Leu Phe Ser Val 35 40 45 Ala Ala Arg Leu Tyr Lys His Pro SerIle Arg Asn Ser Val Ser Leu 50 55 60 Val Val Val Lys Ile Leu Val Ile HisAsp Glu Gln Lys Gly Pro Glu 65 70 75 80 Val Thr Ser Asn Ala Ala Leu ThrLeu Arg Asn Phe Cys Asn Trp Gln 85 90 95 Lys Gln His Asn Pro Pro Ser AspArg Asp Ala Glu His Tyr Asp Thr 100 105 110 Ala Ile Leu Phe Thr Arg GlnAsp Leu Cys Gly Ser Gln Thr Cys Asp 115 120 125 Thr Leu Gly Met Ala AspVal Gly Thr Val Cys Asp Pro Ser Arg Ser 130 135 140 Cys Ser Val Ile GluAsp Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala 145 150 155 160 His Glu LeuGly His Val Phe Asn Met Pro His Asp Asp Ala Lys Gln 165 170 175 Cys AlaSer Leu Asn Gly Val Asn Gln Asp Ser His Met Met Ala Ser 180 185 190 MetLeu Ser Asn Leu Asp His Ser Gln Pro Trp Ser Pro Cys Ser Ala 195 200 205Tyr Met Ile Thr Ser Phe Leu Asp Asn Gly His Gly Glu Cys Leu Met 210 215220 Asp Lys Pro Gln Asn Pro Ile Gln Leu Pro Gly Asp Leu Pro Gly Thr 225230 235 240 Leu Tyr Asp Ala Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu AspSer 245 250 255 Lys His Cys Pro Asp Ala Ala Ser Thr Cys Ser Thr Leu TrpCys Thr 260 265 270 Gly Thr Ser Gly Gly Val Leu Val Cys Gln Thr Lys HisPhe Pro Trp 275 280 285 Ala Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp CysIle Asn Gly Lys 290 295 300 Cys Val Asn Lys Thr Asp Arg Lys His Phe AspThr Pro Phe His Gly 305 310 315 320 Ser Trp Gly Pro Trp Gly Pro Trp GlyAsp Cys Ser Arg Thr Cys Gly 325 330 335 Gly Gly Val Gln Tyr Thr Met ArgGlu Cys Asp Asn Pro Val Pro Lys 340 345 350 Asn Gly Gly Lys Tyr Cys GluGly Lys Arg Val Arg Tyr Arg Ser Cys 355 360 365 Asn Leu Glu Asp Cys ProAsp Asn Asn Gly Lys Thr Phe Arg Glu Glu 370 375 380 Gln Cys Glu Ala HisAsn Glu Phe Ser Lys Ala Ser Phe Gly Ser Gly 385 390 395 400 Pro Ala ValGlu Trp Ile Pro Lys Tyr Ala Gly Val Ser Pro Lys Asp 405 410 415 Arg CysLys Leu Ile Cys Gln Ala Lys Gly Ile Gly Tyr Phe Phe Val 420 425 430 LeuGln Pro Lys Val Val Asp Gly Thr Pro Cys Ser Pro Asp Ser Thr 435 440 445Ser Val Cys Val Gln Gly Gln Cys Val Lys Ala Gly Cys Asp Arg Ile 450 455460 Ile Asp Ser Lys Lys Lys Phe Asp Lys Cys Gly Val Cys Gly Gly Asn 465470 475 480 Gly Ser Thr Cys Lys Lys Ile Ser Gly Ser Val Thr Ser Ala LysPro 485 490 495 Gly Tyr His Asp Ile Val Thr Ile Pro Thr Gly Ala Thr AsnIle Glu 500 505 510 Val Lys Gln Arg Asn Gln Arg Gly Ser Arg Asn Asn GlySer Phe Leu 515 520 525 Ala Ile Lys Ala Ala Asp Gly Thr Tyr Ile Leu AsnGly Asp Tyr Thr 530 535 540 Leu Ser Thr Leu Glu Gln Asp Ile Met Tyr LysGly Val Val Leu Arg 545 550 555 560 Tyr Ser Gly Ser Ser Ala Ala Leu GluArg Ile Arg Ser Phe Ser Pro 565 570 575 Leu Lys Glu Pro Leu Thr Ile GlnVal Leu Thr Val Gly Asn Ala Leu 580 585 590 Arg Pro Lys Ile Lys Tyr ThrTyr Phe Val Lys Lys Lys Lys Glu Ser 595 600 605 Phe Asn Ala Ile Pro ThrPhe Ser Ala Trp Val Ile Glu Glu Trp Gly 610 615 620 Glu Cys Ser Lys SerCys Glu Leu Gly Trp Gln Arg Arg Leu Val Glu 625 630 635 640 Cys Arg AspIle Asn Gly Gln Pro Ala Ser Glu Cys Ala Lys Glu Val 645 650 655 Lys ProAla Ser Thr Arg Pro Cys Ala Asp His Pro Cys Pro Gln Trp 660 665 670 GlnLeu Gly Glu Trp Ser Ser Cys Ser Lys Thr Cys Gly Lys Gly Tyr 675 680 685Lys Lys Arg Ser Leu Lys Cys Leu Ser His Asp Gly Gly Val Leu Ser 690 695700 His Glu Ser Cys Asp Pro Leu Lys Lys Pro Lys His Phe Ile Asp Phe 705710 715 720 Cys Thr Leu Thr Gln Cys Ser 725 2 2184 DNA Homo sapiens exon(1)..(2184) 2 atg gat atc tgc aga att cgg ctt agg aag aag cga ttt gtgtcc agc 48 Met Asp Ile Cys Arg Ile Arg Leu Arg Lys Lys Arg Phe Val SerSer 1 5 10 15 ccc cgt tat gtg gaa acc atg ctt gtg gca gac cag tcg atggca gaa 96 Pro Arg Tyr Val Glu Thr Met Leu Val Ala Asp Gln Ser Met AlaGlu 20 25 30 ttc cac ggc agt ggt cta aag cat tac ctt ctc acg ttg ttt tcggtg 144 Phe His Gly Ser Gly Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val35 40 45 gca gcc aga ttg tac aaa cac ccc agc att cgt aat tca gtt agc ctg192 Ala Ala Arg Leu Tyr Lys His Pro Ser Ile Arg Asn Ser Val Ser Leu 5055 60 gtg gtg gtg aag atc ttg gtc atc cac gat gaa cag aag ggg ccg gaa240 Val Val Val Lys Ile Leu Val Ile His Asp Glu Gln Lys Gly Pro Glu 6570 75 80 gtg acc tcc aat gct gcc ctc act ctg cgg aac ttt tgc aac tgg cag288 Val Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp Gln 8590 95 aag cag cac aac cca ccc agt gac cgg gat gca gag cac tat gac aca336 Lys Gln His Asn Pro Pro Ser Asp Arg Asp Ala Glu His Tyr Asp Thr 100105 110 gca att ctt ttc acc aga cag gac ttg tgt ggg tcc cag aca tgt gat384 Ala Ile Leu Phe Thr Arg Gln Asp Leu Cys Gly Ser Gln Thr Cys Asp 115120 125 act ctt ggg atg gct gat gtt gga act gtg tgt gat ccg agc aga agc432 Thr Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser 130135 140 tgc tcc gtc ata gaa gat gat ggt tta caa gct gcc ttc acc aca gcc480 Cys Ser Val Ile Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala 145150 155 160 cat gaa tta ggc cac gtg ttt aac atg cca cat gat gat gca aagcag 528 His Glu Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala Lys Gln165 170 175 tgt gcc agc ctt aat ggt gtg aac cag gat tcc cac atg atg gcgtca 576 Cys Ala Ser Leu Asn Gly Val Asn Gln Asp Ser His Met Met Ala Ser180 185 190 atg ctt tcc aac ctg gac cac agc cag cct tgg tct cct tgc agtgcc 624 Met Leu Ser Asn Leu Asp His Ser Gln Pro Trp Ser Pro Cys Ser Ala195 200 205 tac atg att aca tca ttt ctg gat aat ggt cat ggg gaa tgt ttgatg 672 Tyr Met Ile Thr Ser Phe Leu Asp Asn Gly His Gly Glu Cys Leu Met210 215 220 gac aag cct cag aat ccc ata cag ctc cca ggc gat ctc cct ggcacc 720 Asp Lys Pro Gln Asn Pro Ile Gln Leu Pro Gly Asp Leu Pro Gly Thr225 230 235 240 ttg tac gat gcc aac cgg cag tgc cag ttt aca ttt ggg gaggac tcc 768 Leu Tyr Asp Ala Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu AspSer 245 250 255 aaa cac tgc ccc gat gca gcc agc aca tgt agc acc ttg tggtgt acc 816 Lys His Cys Pro Asp Ala Ala Ser Thr Cys Ser Thr Leu Trp CysThr 260 265 270 ggc acc tct ggt ggg gtg ctg gtg tgt caa acc aaa cac ttcccg tgg 864 Gly Thr Ser Gly Gly Val Leu Val Cys Gln Thr Lys His Phe ProTrp 275 280 285 gcg gat ggc acc agc tgt gga gaa ggg aaa tgg tgt atc aacggc aag 912 Ala Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp Cys Ile Asn GlyLys 290 295 300 tgt gtg aac aaa acc gac agg aag cat ttt gat acg cct tttcat gga 960 Cys Val Asn Lys Thr Asp Arg Lys His Phe Asp Thr Pro Phe HisGly 305 310 315 320 agc tgg gga cca tgg gga ccg tgg gga gac tgt tcg agaacg tgc ggt 1008 Ser Trp Gly Pro Trp Gly Pro Trp Gly Asp Cys Ser Arg ThrCys Gly 325 330 335 gga gga gtc cag tac acg atg agg gaa tgt gac aac ccagtc cca aag 1056 Gly Gly Val Gln Tyr Thr Met Arg Glu Cys Asp Asn Pro ValPro Lys 340 345 350 aat gga ggg aag tac tgt gaa ggc aaa cga gtg cgc tacaga tcc tgt 1104 Asn Gly Gly Lys Tyr Cys Glu Gly Lys Arg Val Arg Tyr ArgSer Cys 355 360 365 aac ctt gag gac tgt cca gac aat aat gga aaa acc tttaga gag gaa 1152 Asn Leu Glu Asp Cys Pro Asp Asn Asn Gly Lys Thr Phe ArgGlu Glu 370 375 380 caa tgt gaa gca cac aac gag ttt tca aaa gct tcc tttggg agt ggg 1200 Gln Cys Glu Ala His Asn Glu Phe Ser Lys Ala Ser Phe GlySer Gly 385 390 395 400 cct gcg gtg gaa tgg att ccc aag tac gct ggc gtctca cca aag gac 1248 Pro Ala Val Glu Trp Ile Pro Lys Tyr Ala Gly Val SerPro Lys Asp 405 410 415 agg tgc aag ctc atc tgc caa gcc aaa ggc att ggctac ttc ttc gtt 1296 Arg Cys Lys Leu Ile Cys Gln Ala Lys Gly Ile Gly TyrPhe Phe Val 420 425 430 ttg cag ccc aag gtt gtt gat ggt act cca tgt agccca gat tcc acc 1344 Leu Gln Pro Lys Val Val Asp Gly Thr Pro Cys Ser ProAsp Ser Thr 435 440 445 tct gtc tgt gtg caa gga cag tgt gta aaa gct ggttgt gat cgc atc 1392 Ser Val Cys Val Gln Gly Gln Cys Val Lys Ala Gly CysAsp Arg Ile 450 455 460 ata gac tcc aaa aag aag ttt gat aaa tgt ggt gtttgc ggg gga aat 1440 Ile Asp Ser Lys Lys Lys Phe Asp Lys Cys Gly Val CysGly Gly Asn 465 470 475 480 gga tct act tgt aaa aaa ata tca gga tca gttact agt gca aaa cct 1488 Gly Ser Thr Cys Lys Lys Ile Ser Gly Ser Val ThrSer Ala Lys Pro 485 490 495 gga tat cat gat atc gtc aca att cca act ggagcc acc aac atc gaa 1536 Gly Tyr His Asp Ile Val Thr Ile Pro Thr Gly AlaThr Asn Ile Glu 500 505 510 gtg aaa cag cgg aac cag agg gga tcc agg aacaat ggc agc ttt ctt 1584 Val Lys Gln Arg Asn Gln Arg Gly Ser Arg Asn AsnGly Ser Phe Leu 515 520 525 gcc atc aaa gct gct gat ggc aca tat att cttaat ggt gac tac act 1632 Ala Ile Lys Ala Ala Asp Gly Thr Tyr Ile Leu AsnGly Asp Tyr Thr 530 535 540 ttg tcc acc tta gag caa gac att atg tac aaaggt gtt gtc ttg agg 1680 Leu Ser Thr Leu Glu Gln Asp Ile Met Tyr Lys GlyVal Val Leu Arg 545 550 555 560 tac agc ggc tcc tct gcg gca ttg gaa agaatt cgc agc ttt agc cct 1728 Tyr Ser Gly Ser Ser Ala Ala Leu Glu Arg IleArg Ser Phe Ser Pro 565 570 575 ctc aaa gag ccc ttg acc atc cag gtt cttact gtg ggc aat gcc ctt 1776 Leu Lys Glu Pro Leu Thr Ile Gln Val Leu ThrVal Gly Asn Ala Leu 580 585 590 cga cct aaa att aaa tac acc tac ttc gtaaag aag aag aag gaa tct 1824 Arg Pro Lys Ile Lys Tyr Thr Tyr Phe Val LysLys Lys Lys Glu Ser 595 600 605 ttc aat gct atc ccc act ttt tca gca tgggtc att gaa gag tgg ggc 1872 Phe Asn Ala Ile Pro Thr Phe Ser Ala Trp ValIle Glu Glu Trp Gly 610 615 620 gaa tgt tct aag tca tgt gaa ttg ggt tggcag aga aga ctg gta gaa 1920 Glu Cys Ser Lys Ser Cys Glu Leu Gly Trp GlnArg Arg Leu Val Glu 625 630 635 640 tgc cga gac att aat gga cag cct gcttcc gag tgt gca aag gaa gtg 1968 Cys Arg Asp Ile Asn Gly Gln Pro Ala SerGlu Cys Ala Lys Glu Val 645 650 655 aag cca gcc agc acc aga cct tgt gcagac cat ccc tgc ccc cag tgg 2016 Lys Pro Ala Ser Thr Arg Pro Cys Ala AspHis Pro Cys Pro Gln Trp 660 665 670 cag ctg ggg gag tgg tca tca tgt tctaag acc tgt ggg aag ggt tac 2064 Gln Leu Gly Glu Trp Ser Ser Cys Ser LysThr Cys Gly Lys Gly Tyr 675 680 685 aaa aaa aga agc ttg aag tgt ctg tcccat gat gga ggg gtg tta tct 2112 Lys Lys Arg Ser Leu Lys Cys Leu Ser HisAsp Gly Gly Val Leu Ser 690 695 700 cat gag agc tgt gat cct tta aag aaacct aaa cat ttc ata gac ttt 2160 His Glu Ser Cys Asp Pro Leu Lys Lys ProLys His Phe Ile Asp Phe 705 710 715 720 tgc aca ctg aca cag tgc agt taa2184 Cys Thr Leu Thr Gln Cys Ser 725 3 10 PRT Mus sp. 3 Arg Thr Cys GlyGly Gly Val Gln Tyr Thr 1 5 10 4 30 DNA Mus sp. 4 agaacctgtg gtggtggagttcaatacaca 30 5 32 DNA Mus sp. 5 cctcttaact gcactgtgtc agtgtgcaaa ag 326 23 DNA Homo sapiens 6 cctcttaact gcactgtgtc agt 23 7 24 DNA Homosapiens 7 caggcccact cccaaaggaa gctt 24 8 27 DNA Artificial Sequence AP1primer 8 ccatcctaat acgactcact atagggc 27 9 23 DNA Artificial SequenceAP2 primer 9 actcactata gggctcgagc ggc 23 10 43 DNA Artificial SequenceForward Primer (2) 10 caccccggga ggaagaagcg atttgtgtcc agcccccgtt atg 4311 42 DNA Artificial Sequence Back primer (2) 11 gtggcggccg ccctcttaactgcactgtgt cagtgtgcaa aa 42 12 727 PRT Mus sp. 12 Glu Pro Ser Gly ProGly Ser Ile Arg Lys Lys Arg Phe Val Ser Ser 1 5 10 15 Pro Arg Tyr ValGlu Thr Met Leu Val Ala Asp Gln Ser Met Ala Asp 20 25 30 Phe His Gly SerGly Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val 35 40 45 Ala Ala Arg PheTyr Lys His Pro Ser Ile Arg Asn Ser Ile Ser Leu 50 55 60 Val Val Val LysIle Leu Val Ile Tyr Glu Glu Gln Lys Gly Pro Glu 65 70 75 80 Val Thr SerAsn Ala Ala Leu Thr Leu Arg Asn Phe Cys Ser Trp Gln 85 90 95 Lys Gln HisAsn Ser Pro Ser Asp Arg Asp Pro Glu His Tyr Asp Thr 100 105 110 Ala IleLeu Phe Thr Arg Gln Asp Leu Cys Gly Ser His Thr Cys Asp 115 120 125 ThrLeu Gly Met Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser 130 135 140Cys Ser Val Ile Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala 145 150155 160 His Glu Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala Lys His165 170 175 Cys Ala Ser Leu Asn Gly Val Ser Gly Asp Ser His Leu Met AlaSer 180 185 190 Met Leu Ser Ser Leu Asp His Ser Gln Pro Trp Ser Pro CysSer Ala 195 200 205 Tyr Met Val Thr Ser Phe Leu Asp Asn Gly His Gly GluCys Leu Met 210 215 220 Asp Lys Pro Gln Asn Pro Ile Lys Leu Pro Ser AspLeu Pro Gly Thr 225 230 235 240 Leu Tyr Asp Ala Asn Arg Gln Cys Gln PheThr Phe Gly Glu Glu Ser 245 250 255 Lys His Cys Pro Asp Ala Ala Ser ThrCys Thr Thr Leu Trp Cys Thr 260 265 270 Gly Thr Ser Gly Gly Leu Leu ValCys Gln Thr Lys His Phe Pro Trp 275 280 285 Ala Asp Gly Thr Ser Cys GlyGlu Gly Lys Trp Cys Val Ser Gly Lys 290 295 300 Cys Val Asn Lys Thr AspMet Lys His Phe Ala Thr Pro Val His Gly 305 310 315 320 Ser Trp Gly ProTrp Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys Gly 325 330 335 Gly Gly ValGln Tyr Thr Met Arg Glu Cys Asp Asn Pro Val Pro Lys 340 345 350 Asn GlyGly Lys Tyr Cys Glu Gly Lys Arg Val Arg Tyr Arg Ser Cys 355 360 365 AsnIle Glu Asp Cys Pro Asp Asn Asn Gly Lys Thr Phe Arg Glu Glu 370 375 380Gln Cys Glu Ala His Asn Glu Phe Ser Lys Ala Ser Phe Gly Asn Glu 385 390395 400 Pro Thr Val Glu Trp Thr Pro Lys Tyr Ala Gly Val Ser Pro Lys Asp405 410 415 Arg Cys Lys Leu Thr Cys Glu Ala Lys Gly Ile Gly Tyr Phe PheVal 420 425 430 Leu Gln Pro Lys Val Val Asp Gly Thr Pro Cys Ser Pro AspSer Thr 435 440 445 Ser Val Cys Val Gln Gly Gln Cys Val Lys Ala Gly CysAsp Arg Ile 450 455 460 Ile Asp Ser Lys Lys Lys Phe Asp Lys Cys Gly ValCys Gly Gly Asn 465 470 475 480 Gly Ser Thr Cys Lys Lys Met Ser Gly IleVal Thr Ser Thr Arg Pro 485 490 495 Gly Tyr His Asp Ile Val Thr Ile ProAla Gly Ala Thr Asn Ile Glu 500 505 510 Val Lys His Arg Asn Gln Arg GlySer Arg Asn Asn Gly Ser Phe Leu 515 520 525 Ala Ile Arg Ala Ala Asp GlyThr Tyr Ile Leu Asn Gly Asn Phe Thr 530 535 540 Leu Ser Thr Leu Glu GlnAsp Leu Thr Tyr Lys Gly Thr Val Leu Arg 545 550 555 560 Tyr Ser Gly SerSer Ala Ala Leu Glu Arg Ile Arg Ser Phe Ser Pro 565 570 575 Leu Lys GluPro Leu Thr Ile Gln Val Leu Met Val Gly His Ala Leu 580 585 590 Arg ProLys Ile Lys Phe Thr Tyr Phe Met Lys Lys Lys Thr Glu Ser 595 600 605 PheAsn Ala Ile Pro Thr Phe Ser Glu Trp Val Ile Glu Glu Trp Gly 610 615 620Glu Cys Ser Lys Thr Cys Gly Ser Gly Trp Gln Arg Arg Val Val Gln 625 630635 640 Cys Arg Asp Ile Asn Gly His Pro Ala Ser Glu Cys Ala Lys Glu Val645 650 655 Lys Pro Ala Ser Thr Arg Pro Cys Ala Asp Leu Pro Cys Pro HisTrp 660 665 670 Gln Val Gly Asp Trp Ser Pro Cys Ser Lys Thr Cys Gly LysGly Tyr 675 680 685 Lys Lys Arg Thr Leu Lys Cys Val Ser His Asp Gly GlyVal Leu Ser 690 695 700 Asn Glu Ser Cys Asp Pro Leu Lys Lys Pro Lys HisTyr Ile Asp Phe 705 710 715 720 Cys Thr Leu Thr Gln Cys Ser 725 13 2184DNA Mus sp. exon (1)..(2184) 13 aag cca tca gga cca gga agc ata agg aagaag cga ttt gtg tcc agc 48 Lys Pro Ser Gly Pro Gly Ser Ile Arg Lys LysArg Phe Val Ser Ser 1 5 10 15 ccc cgt tat gtg gaa acc atg ctc gta gctgac cag tcc atg gcc gac 96 Pro Arg Tyr Val Glu Thr Met Leu Val Ala AspGln Ser Met Ala Asp 20 25 30 ttc cac ggc agc ggt cta aag cat tac ctt ctaacc ctg ttc tcg gtg 144 Phe His Gly Ser Gly Leu Lys His Tyr Leu Leu ThrLeu Phe Ser Val 35 40 45 gca gcc agg ttt tac aag cat ccc agc att agg aattca att agc ctg 192 Ala Ala Arg Phe Tyr Lys His Pro Ser Ile Arg Asn SerIle Ser Leu 50 55 60 gtg gtg gtg aag atc ttg gtc ata tac gag gag cag aaggga cca gaa 240 Val Val Val Lys Ile Leu Val Ile Tyr Glu Glu Gln Lys GlyPro Glu 65 70 75 80 gtt acc tcc aat gca gct ctc acc ctt cgg aat ttc tgcagc tgg cag 288 Val Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys SerTrp Gln 85 90 95 aaa caa cac aac agc ccc agt gac cgg gat cca gag cac tatgac act 336 Lys Gln His Asn Ser Pro Ser Asp Arg Asp Pro Glu His Tyr AspThr 100 105 110 gca att ctg ttc acc aga cag gat tta tgt ggc tcc cac acgtgt gac 384 Ala Ile Leu Phe Thr Arg Gln Asp Leu Cys Gly Ser His Thr CysAsp 115 120 125 act ctc gga atg gca gat gtt gga acc gta tgt gac ccc agcagg agc 432 Thr Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp Pro Ser ArgSer 130 135 140 tgc tca gtc ata gaa gat gat ggt ttg caa gct gcc ttc accaca gcc 480 Cys Ser Val Ile Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr ThrAla 145 150 155 160 cat gaa ttg ggc cat gtg ttt aac atg ccg cac gat gatgct aag cac 528 His Glu Leu Gly His Val Phe Asn Met Pro His Asp Asp AlaLys His 165 170 175 tgt gcc agc ttg aat ggt gtg agt ggc gat tct cat ctgatg gcc tcg 576 Cys Ala Ser Leu Asn Gly Val Ser Gly Asp Ser His Leu MetAla Ser 180 185 190 atg ctc tcc agc tta gac cat agc cag ccc tgg tca ccttgc agt gcc 624 Met Leu Ser Ser Leu Asp His Ser Gln Pro Trp Ser Pro CysSer Ala 195 200 205 tac atg gtc acg tcc ttc cta gat aat gga cac ggg gaatgt ttg atg 672 Tyr Met Val Thr Ser Phe Leu Asp Asn Gly His Gly Glu CysLeu Met 210 215 220 gac aag ccc cag aat cca atc aag ctc cct tct gat cttccc ggt acc 720 Asp Lys Pro Gln Asn Pro Ile Lys Leu Pro Ser Asp Leu ProGly Thr 225 230 235 240 ttg tac gat gcc aac cgc cag tgt cag ttt aca ttcgga gag gaa tcc 768 Leu Tyr Asp Ala Asn Arg Gln Cys Gln Phe Thr Phe GlyGlu Glu Ser 245 250 255 aag cac tgc cct gat gca gcc agc aca tgt act accctg tgg tgc act 816 Lys His Cys Pro Asp Ala Ala Ser Thr Cys Thr Thr LeuTrp Cys Thr 260 265 270 ggc acc tcc ggt ggc tta ctg gtg tgc caa aca aaacac ttc cct tgg 864 Gly Thr Ser Gly Gly Leu Leu Val Cys Gln Thr Lys HisPhe Pro Trp 275 280 285 gca gat ggc acc agc tgt gga gaa ggg aag tgg tgtgtc agt ggc aag 912 Ala Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp Cys ValSer Gly Lys 290 295 300 tgc gtg aac aag aca gac atg aag cat ttt gct actcct gtt cat gga 960 Cys Val Asn Lys Thr Asp Met Lys His Phe Ala Thr ProVal His Gly 305 310 315 320 agc tgg gga cca tgg gga ccg tgg gga gac tgctca aga acc tgt ggt 1008 Ser Trp Gly Pro Trp Gly Pro Trp Gly Asp Cys SerArg Thr Cys Gly 325 330 335 ggt gga gtt caa tac aca atg aga gaa tgt gacaac cca gtc cca aag 1056 Gly Gly Val Gln Tyr Thr Met Arg Glu Cys Asp AsnPro Val Pro Lys 340 345 350 aac gga ggg aag tac tgt gaa ggc aaa cga gtccgc tac agg tcc tgt 1104 Asn Gly Gly Lys Tyr Cys Glu Gly Lys Arg Val ArgTyr Arg Ser Cys 355 360 365 aac atc gag gac tgt cca gac aat aac gga aaaacg ttc aga gag gag 1152 Asn Ile Glu Asp Cys Pro Asp Asn Asn Gly Lys ThrPhe Arg Glu Glu 370 375 380 cag tgc gag gcg cac aat gag ttt tcc aaa gcttcc ttt ggg aat gag 1200 Gln Cys Glu Ala His Asn Glu Phe Ser Lys Ala SerPhe Gly Asn Glu 385 390 395 400 ccc act gta gag tgg aca ccc aag tac gccggc gtc tcg cca aag gac 1248 Pro Thr Val Glu Trp Thr Pro Lys Tyr Ala GlyVal Ser Pro Lys Asp 405 410 415 agg tgc aag ctc acc tgt gaa gcc aaa ggcatt ggc tac ttt ttc gtc 1296 Arg Cys Lys Leu Thr Cys Glu Ala Lys Gly IleGly Tyr Phe Phe Val 420 425 430 tta cag ccc aag gtt gta gat ggc act ccctgt agt cca gac tct acc 1344 Leu Gln Pro Lys Val Val Asp Gly Thr Pro CysSer Pro Asp Ser Thr 435 440 445 tct gtc tgt gtg caa ggg cag tgt gtg aaagct ggc tgt gat cgc atc 1392 Ser Val Cys Val Gln Gly Gln Cys Val Lys AlaGly Cys Asp Arg Ile 450 455 460 ata gac tcc aaa aag aag ttt gat aag tgtggc gtt tgt gga gga aac 1440 Ile Asp Ser Lys Lys Lys Phe Asp Lys Cys GlyVal Cys Gly Gly Asn 465 470 475 480 ggt tcc aca tgc aag aag atg tca ggaata gtc act agt aca aga cct 1488 Gly Ser Thr Cys Lys Lys Met Ser Gly IleVal Thr Ser Thr Arg Pro 485 490 495 ggg tat cat gac att gtc aca att cctgct gga gcc acc aac att gaa 1536 Gly Tyr His Asp Ile Val Thr Ile Pro AlaGly Ala Thr Asn Ile Glu 500 505 510 gtg aaa cat cgg aat caa agg ggg tccaga aac aat ggc agc ttt ctg 1584 Val Lys His Arg Asn Gln Arg Gly Ser ArgAsn Asn Gly Ser Phe Leu 515 520 525 gct att aga gcc gct gat ggt acc tatatt ctg aat gga aac ttc act 1632 Ala Ile Arg Ala Ala Asp Gly Thr Tyr IleLeu Asn Gly Asn Phe Thr 530 535 540 ctg tcc aca cta gag caa gac ctc acctac aaa ggt act gtc tta agg 1680 Leu Ser Thr Leu Glu Gln Asp Leu Thr TyrLys Gly Thr Val Leu Arg 545 550 555 560 tac agt ggt tcc tcg gct gcg ctggaa aga atc cgc agc ttt agt cca 1728 Tyr Ser Gly Ser Ser Ala Ala Leu GluArg Ile Arg Ser Phe Ser Pro 565 570 575 ctc aaa gaa ccc tta acc atc caggtt ctt atg gta ggc cat gct ctc 1776 Leu Lys Glu Pro Leu Thr Ile Gln ValLeu Met Val Gly His Ala Leu 580 585 590 cga ccc aaa att aaa ttc acc tacttt atg aag aag aag aca gag tca 1824 Arg Pro Lys Ile Lys Phe Thr Tyr PheMet Lys Lys Lys Thr Glu Ser 595 600 605 ttc aac gcc att ccc aca ttt tctgag tgg gtg att gaa gag tgg ggg 1872 Phe Asn Ala Ile Pro Thr Phe Ser GluTrp Val Ile Glu Glu Trp Gly 610 615 620 gag tgc tcc aag aca tgc ggc tcaggt tgg cag aga aga gta gtg cag 1920 Glu Cys Ser Lys Thr Cys Gly Ser GlyTrp Gln Arg Arg Val Val Gln 625 630 635 640 tgc aga gac att aac gga caccct gct tcc gaa tgt gca aag gaa gtg 1968 Cys Arg Asp Ile Asn Gly His ProAla Ser Glu Cys Ala Lys Glu Val 645 650 655 aag cca gcc agt acc aga ccttgt gca gac ctt cct tgc cca cac tgg 2016 Lys Pro Ala Ser Thr Arg Pro CysAla Asp Leu Pro Cys Pro His Trp 660 665 670 cag gtg ggg gat tgg tca ccatgt tcc aaa act tgc ggg aag ggt tac 2064 Gln Val Gly Asp Trp Ser Pro CysSer Lys Thr Cys Gly Lys Gly Tyr 675 680 685 aag aag aga acc ttg aaa tgtgtg tcc cac gat ggg ggc ctg tta tca 2112 Lys Lys Arg Thr Leu Lys Cys ValSer His Asp Gly Gly Leu Leu Ser 690 695 700 aat gag agc tgt gat cct ttgaag aag cca aag cat tac att gac ttt 2160 Asn Glu Ser Cys Asp Pro Leu LysLys Pro Lys His Tyr Ile Asp Phe 705 710 715 720 tgc aca ctg aca cag tgcagt taa 2184 Cys Thr Leu Thr Gln Cys Ser 725 14 303 DNA Homo sapiens 14caagaacctg tggtggtgga gttcattaca cgatgaggga ttgttacaac ccagtcccaa 60agattggagg gaagtcttgt aaaggcaaac gagtgcccta cagttccttt atcctttagg 120actgtctaga caattactgg aattcgactt aagagtggcc catcctatgc gccacaccgc 180gtttcaaaat gtttcctttg ggagttgggc tgcggtggaa ttggttttcc caaggatcgt 240ggcgtctcac caaaggacag gtgcaagctc atcatgccaa gccaaaggat tggctacatt 300ttc 303

1. A protein characterized by containing an amino acid sequence of SEQID NO: 1: Met Asp Ile Cys Arg Ile Arg Leu Arg Lys Lys Arg Phe Val SerSer Pro Arg Tyr Val Glu Thr Met Leu Val Ala Asp Gln Ser Met Ala Glu PheHis Gly Ser Gly Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val Ala Ala ArgLeu Tyr Lys His Pro Ser Ile Arg Asn Ser Val Ser Leu Val Val Val Lys IleLeu Val Ile His Asp Glu Gln Lys Gly Pro Glu Val Thr Ser Asn Ala Ala LeuThr Leu Arg Asn Phe Cys Asn Trp Gln Lys Gln His Asn Pro Pro Ser Asp ArgAsp Ala Glu His Tyr Asp Thr Ala Ile Leu Phe Thr Arg Gln Asp Leu Cys GlySer Gln Thr Cys Asp Thr Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp ProSer Arg Ser Cys Ser Val Ile Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr ThrAla His Glu Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala Lys Gln CysAla Ser Leu Asn Gly Val Asn Gln Asp Ser His Met Met Ala Ser Met Leu SerAsn Leu Asp His Ser Gln Pro Trp Ser Pro Cys Ser Ala Tyr Met Ile Thr SerPhe Leu Asp Asn Gly His Gly Glu Cys Leu Met Asp Lys Pro Gln Asn Pro IleGln Leu Pro Gly Asp Leu Pro Gly Thr Leu Tyr Asp Ala Asn Arg Gln Cys GlnPhe Thr Phe Gly Glu Asp Ser Lys His Cys Pro Asp Ala Ala Ser Thr Cys SerThr Leu Trp Cys Thr Gly Thr Ser Gly Gly Val Leu Val Cys Gln Thr Lys HisPhe Pro Trp Ala Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp Cys Ile Asn GlyLys Cys Val Asn Lys Thr Asp Arg Lys His Phe Asp Thr Pro Phe His Gly SerTrp Gly Pro Trp Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys Gly Gly Gly ValGln Tyr Thr Met Arg Glu Cys Asp Asn Pro Val Pro Lys Asn Gly Gly Lys TyrCys Glu Gly Lys Arg Val Arg Tyr Arg Ser Cys Asn Leu Glu Asp Cys Pro AspAsn Asn Gly Lys Thr Phe Arg Glu Glu Gln Cys Glu Ala His Asn Glu Phe SerLys Ala Ser Phe Gly Ser Gly Pro Ala Val Glu Trp Ile Pro Lys Tyr Ala GlyVal Ser Pro Lys Asp Arg Cys Lys Leu Ile Cys Gln Ala Lys Gly Ile Gly TyrPhe Phe Val Leu Gln Pro Lys Val Val Asp Gly Thr Pro Cys Ser Pro Asp SerThr Ser Val Cys Val Gln Gly Gln Cys Val Lys Ala Gly Cys Asp Arg Ile IleAsp Ser Lys Lys Lys Phe Asp Lys Cys Gly Val Cys Gly Gly Asn Gly Ser ThrCys Lys Lys Ile Ser Gly Ser Val Thr Ser Ala Lys Pro Gly Tyr His Asp IleVal Thr Ile Pro Thr Gly Ala Thr Asn Ile Glu Val Lys Gln Arg Asn Gln ArgGly Ser Arg Asn Asn Gly Ser Phe Leu Ala Ile Lys Ala Ala Asp Gly Thr TyrIle Leu Asn Gly Asp Tyr Thr Leu Ser Thr Leu Glu Gln Asp Ile Met Tyr LysGly Val Val Leu Arg Tyr Ser Gly Ser Ser Ala Ala Leu Glu Arg Ile Arg SerPhe Ser Pro Leu Lys Glu Pro Leu Thr Ile Gln Val Leu Thr Val Gly Asn AlaLeu Arg Pro Lys Ile Lys Tyr Thr Tyr Phe Val Lys Lys Lys Lys Glu Ser PheAsn Ala Ile Pro Thr Phe Ser Ala Trp Val Ile Glu Glu Trp Gly Glu Cys SerLys Ser Cys Glu Leu Gly Trp Gln Arg Arg Leu Val Glu Cys Arg Asp Ile AsnGly Gln Pro Ala Ser Glu Cys Ala Lys Glu Val Lys Pro Ala Ser Thr Arg ProCys Ala Asp His Pro Cys Pro Gln Trp Gln Leu Gly Glu Trp Ser Ser Cys SerLys Thr Cys Gly Lys Gly Tyr Lys Lys Arg Ser Leu Lys Cys Leu Ser His AspGly Gly Val Leu Ser His Glu Ser Cys Asp Pro Leu Lys Lys Pro Lys His PheIle Asp Phe Cys Thr Leu Thr Gln Cys Ser.


2. A variation functionally equivalent to a protein containing an aminoacid sequence of SEQ ID NO:
 1. 3. A protein characterized by containinga matrix metalloproteinase domain, a disintegrin domain, and athrombospondin domain, except for a mouse ADAMTS-1 protein.
 4. A genecharacterized by encoding said protein according to any one of claims 1to
 3. 5. A gene containing a base sequence of SEQ ID NO: 2: ATG GAT ATCTGC AGA ATT CGG CTT AGG AAG AAG CGA TTT GTG TCC AGC CCC CGT TAT GTG GAAACC ATG CTT GTG GCA GAC CAG TCG ATG GCA GAA TTC CAC GGC AGT GGT CTA AAGCAT TAC CTT CTC ACG TTG TTT TCG GTG GCA GCC AGA TTG TAC AAA CAC CCC AGCATT CGT AAT TCA GTT AGC CTG GTG GTG GTG AAG ATC TTG GTC ATC CAC GAT GAACAG AAG GGG CCG GAA GTG ACC TCC AAT GCT GCC CTC ACT CTG CGG AAC TTT TGCAAC TGG CAG AAG CAG CAC AAC CCA CCC AGT GAC CGG GAT GCA GAG CAC TAT GACACA GCA ATT CTT TTC ACC AGA CAG GAC TTG TGT GGG TCC CAG ACA TGT GAT ACTCTT GGG ATG GCT GAT GTT GGA ACT GTG TGT GAT CCG AGC AGA AGC TGC TCC GTCATA GAA GAT GAT GGT TTA CAA GCT GCC TTC ACC ACA GCC CAT GAA TTA GGC CACGTG TTT AAC ATG CCA CAT GAT GAT GCA AAG CAG TGT GCC AGC CTT AAT GGT GTGAAC CAG GAT TCC CAC ATG ATG GCG TCA ATG CTT TCC AAC CTG GAC CAC AGC GAGCCT TGG TCT CCT TGC AGT GCC TAC ATG ATT ACA TCA TTT CTG GAT AAT GGT CATGGG GAA TGT TTG ATG GAC AAG CCT CAG AAT CCC ATA CAG CTC CCA GGC GAT CTCCCT GGC ACC TTG TAC GAT GCC AAC CGG CAG TGC CAG TTT ACA TTT GGG GAG GACTCC AAA CAC TGC CCC GAT GCA GCC AGC ACA TGT AGC ACC TTG TGG TGT ACC GGCACC TCT GGT GGG GTG CTG GTG TGT CAA ACC AAA CAC TTC CCG TGG GCG GAT GGCACC AGC TGT GGA GAA GGG AAA TGG TGT ATC AAC GGC AAG TGT GTG AAC AAA ACCGAC AGG AAG CAT TTT GAT ACG CCT TTT CAT GGA AGC TGC GGA CCA TGG GGA CCGTGG GGA GAC TGT TCG AGA ACG TGC GGT GGA GGA GTC CAG TAC ACG ATG AGG GAATGT GAC AAC CCA GTC CCA AAG AAT GGA GGG AAG TAC TGT GAA GGC AAA CGA GTGCGC TAC AGA TCC TGT AAC CTT GAG GAC TGT CCA GAC AAT AAT GGA AAA ACC TTTAGA GAG GAA CAA TGT GAA GCA CAC AAC GAG TTT TCA AAA CGT TCC TTT GGG AGTGGG CCT GCG GTG GAA TGC ATT CCC AAG TAC GCT GGC GTC TCA CCA AAG GAC AGGTGG AAG CTC ATC TGC CAA GCC AAA GGC ATT GGC TAC TTC TTC GTT TTG CAG CCCAAG GTT GTT GAT GGT ACT CCA TGT AGC CCA GAT TCC ACC TCT GTC TGT GTG CAAGGA CAG TGT GTA AAA GCT GGT TGT GAT CGC ATC ATA GAC TCC AAA AAG AAG TTTGAT AAA TGT GGT GTT TGC GGG GGA AAT GGA TCT ACT TGT AAA AAA ATA TCA GGATCA GTT ACT AGT GCA AAA CCT GGA TAT CAT GAT ATC GTC ACA ATT CCA ACT GGAGCC ACC AAC ATC GAA GTG AAA CAG CGG AAC CAG AGG GGA TCC AGG AAC AAT GGCAGC TTT CTT GCC ATC AAA GCT GCT GAT GGC ACA TAT ATT CTT AAT GGT GAC TACACT TTG TCC ACC TTA GAG CAA GAC ATT ATG TAC AAA GGT GTT GTC TTC AGG TACAGC GGC TCC TCT GCG GCA TTG GAA AGA ATT CGC AGC TTT AGC CCT CTC AAA GAGCCC TTG ACC ATC CAG GTT CTT ACT GTG GGC AAT GCC CTT CGA CCT AAA ATT AAATAC ACC TAC TTC GTA AAG AAG AAG AAG GAA TCT TTC AAT GCT ATC CCC ACT TTTTCA GCA TGG GTC ATT GAA GAG TGG GGC GAA TGT TCT AAG TCA TGT GAA TTG GGTTGG CAG AGA AGA CTG GTA GAA TGC CGA GAC ATT AAT GGA CAG CCT GCT TCC GAGTCT GCA AAG GAA GTG AAG CCA GCC AGC ACC AGA CCT TGT GCA GAC CAT CCC TGCCCC CAG TGG CAG CTG GGG GAG TGG TCA TCA TGT TCT AAG ACC TGT GGG AAG GGTTAC AAA AAA AGA AGC TTG AAG TGT CTG TCC CAT GAT GGA GGG GTG TTA TCT CATGAG AGC TGT GAT CCT TTA AAG AAA CCT AAA CAT TTC ATA GAC TTT TGC ACA CTGACA CAG TGC AGT TAA.


6. A vector characterized by containing said gene according to claim 4or
 5. 7. A transformant characterized by being transformed by saidvector according to claim
 6. 8. A pharmaceutical compositioncharacterized by comprising (1) a protein containing an amino acidsequence of SEQ ID NO: 1, (2) a variation functionally equivalent tosaid protein containing said amino acid sequence of SEQ ID NO: 1, or (3)a protein containing a matrix metalloproteinase domain, a disintegrindomain, and a thrombospondin domain, and a pharmaceutically orveterinarily acceptable carrier.
 9. The pharmaceutical compositionaccording to claim 8, for reducing leukocytes.
 10. The pharmaceuticalcomposition according to claim 8, for reducing platelets.
 11. Thepharmaceutical composition according to claim 8, for increasingerythrocytes.
 12. A food characterized by comprising (1) a proteincontaining an amino acid sequence of SEQ ID NO: 1, (2) a variationfunctionally equivalent to said protein containing said amino acidsequence of SEQ ID NO: 1, or (3) a protein containing a matrixmetalloproteinase domain, a disintegrin domain, and a thrombospondindomain, and a food additive.
 13. A food additive characterized bycomprising (1) a protein containing an amino acid sequence of SEQ ID NO:1, (2) a variation functionally equivalent to said protein containingsaid amino acid sequence of SEQ ID NO: 1, or (3) a protein containing amatrix metalloproteinase domain, a disintegrin domain, and athrombospondin domain.
 14. An immunologically reactive substancecharacterized by being reactive specifically to said protein accordingto any one of claims 1 to
 3. 15. A method for immunologically analyzinga human ADAMTS-1 protein containing an amino acid sequence of SEQ ID NO:1, characterized in that a sample is brought into contact with animmunologically reactive substance which is reactive specifically tosaid human ADAMTS-1 protein, and a complex of said human ADAMTS-1protein and said immunologically reactive substance is detected.
 16. Amethod for extracorporeally detecting an immunological state,characterized by analyzing a human ADAMTS-1 protein containing an aminoacid sequence of SEQ ID NO: 1 in a sample.
 17. A method forimmunologically analyzing an mRNA of a human ADAMTS-1 protein containingan amino acid sequence of SEQ ID NO: 1, characterized in that a sampleis brought into contact with a polynucleotide containing a base sequencecomplementary to that of said mRNA of said human ADAMTS-1 protein, and acomplex of said mRNA of said human ADAMTS-1 protein and said gene isdetected.
 18. A method for extracorporeally detecting an immunologicalstate, characterized by analyzing an mRNA of a human ADAMTS-1 proteincontaining an amino acid sequence of SEQ ID NO: 1 in a sample.
 19. Anagent for analyzing an immunological state, characterized by containingan immunologically reactive substance capable of immunologicallyreacting a human ADAMTS-1 protein containing an amino acid sequence ofSEQ ID NO:
 1. 20. An agent for analyzing an immunological state,characterized by containing a polynucleotide containing a base sequencecomplementary to that of an mRNA of a human ADAMTS-1 protein containingan amino acid sequence of SEQ ID NO:
 1. 21. A method for reducingleukocytes, comprising administering to a subject in need thereof, (1) aprotein containing an amino acid sequence of SEQ ID NO: 1, (2) avariation functionally equivalent to said protein containing said aminoacid sequence of SEQ ID NO: 1, or (3) a protein containing a matrixmetalloproteinase domain, a disintegrin domain, and a thrombospondindomain, in an amount effective therefor.
 22. A method for reducingplatelets, comprising administering to a subject in need thereof, (1) aprotein containing an amino acid sequence of SEQ ID NO: 1, (2) avariation functionally equivalent to said protein containing said aminoacid sequence of SEQ ID NO: 1, or (3) a protein containing a matrixmetalloproteinase domain, a disintegrin domain, and a thrombospondindomain, in an amount effective therefor.
 23. A method for increasingerythrocytes, comprising administering to a subject in need thereof, (1)a protein containing an amino acid sequence of SEQ ID NO: 1, (2) avariation functionally equivalent to said protein containing said aminoacid sequence of SEQ ID NO: 1, or (3) a protein containing a matrixmetalloproteinase domain, a disintegrin domain, and a thrombospondindomain, in an amount effective therefor.
 24. Use of (1) a proteincontaining an amino acid sequence of SEQ ID NO: 1, (2) a variationfunctionally equivalent to said protein containing said amino acidsequence of SEQ ID NO: 1, or (3) a protein containing a matrixmetalloproteinase domain, a disintegrin domain, and a thrombospondindomain, for preparing a pharmaceutical composition.